Calcium Prevents Enhanced Degradation of Factor VIII in the Condition of Motion.

Background: Hemophilia A and B induce recurrent bleeding episodes, mainly in skeletal muscles and joints that are in intermittent motion. We have previously demonstrated that intermittent motion contributes to increased degradation of factors VIII and IX. Objectives: Given that calcium ions are known to enhance factor VIII-von Willebrand factor (vWF) interaction, the present study has investigated the role of these ions on factors VIII and IX in the condition of motion. Methods: The effects of calcium ions were assessed using purified proteins via Western blot, factor VIII activity, immunocytochemistry, and in Institute of Cancer Research (ICR) mice with no specific genetic background. Results: Calcium was found to prevent degradation of plasma-derived factor VIII but not that of factor IX, during intermittent motion. Calcium levels in the microcirculation of mouse striated muscles were elevated following movement, enabling prevention of factor VIII degradation in normal physiology. Calcium supplementation in drinking water increased factor VIII levels in blood and striated muscles of ICR mice during movement. Conclusions: calcium ions decrease factor VIII degradation in the condition of motion. Further research on the impact of calcium salt oral supplementation on hemophilia patients is warranted.

Tissue factor-heparanase complex: intracellular nonhemostatic effects.

Background: Heparanase, known to be involved in angiogenesis, cancer progression, and inflammation, was shown to form a complex with tissue factor (TF) via its procoagulant domain and to enhance the hemostatic system. Objectives: To reveal a potential role of heparanase procoagulant domain in nonhemostatic effects. Methods: Effects of peptides 16 and 16AC derived from the heparanase procoagulant domain, discovered by our group, were studied using the XTT proliferation assay, western blot analysis, and immunostaining in vitro and a mouse wound-healing model. Results: Procoagulant peptides induced increased proliferation, release of heparanase, and upregulation of heparanase, TF, tissue factor pathway inhibitor (TFPI), and TFPI-2 in U87, T47D, and MCF-7 tumor cell lines and in endothelial cells. These results were reversed by a peptide derived from TFPI-2 that inhibited the heparanse procoagulant domain-TF complex. Thrombin had a similar effect on tumor cell proliferation and heparanase release, although the impact of thrombin on cell proliferation was mediated by the heparanase procoagulant domain. A mouse model of full-thickness skin incision exhibited higher levels of heparanase, TF, TFPI, and TFPI-2 in the healing skin, mainly in the blood vessel wall and lumen in animals injected with the procoagulant peptides compared to controls. The cells transfected to overexpress full-length TF or TF devoid of the cytoplasmic domain demonstrated that the procoagulant domain conveyed intracellular signaling via TF. Conclusion: Heparanase procoagulant domain induces nonhemostatic effects via TF. The finding that TF serves as a receptor to heparanase supports the close direct relation between the hemostatic system and cancer progression.

Effects of anti-Xa activity monitoring on the outcome of high-risk pregnancies treated with a prophylactic dose of low-molecular-weight heparin.

OBJECTIVE: To evaluate if anti-Xa level monitoring and dose adjustment in women using a prophylactic dose of enoxaparin can decrease placenta-mediated pregnancy complications. METHODS: This retrospective observational cohort study included pregnant women receiving enoxaparin prophylaxis, who were followed at the Thrombosis and Hemostasis Outpatient clinic between 2010 and 2017. The dose was adjusted according to enoxaparin anti-Xa levels in the study group or the weight of individuals in the control group. RESULTS: Of 585 women surveyed, 110 met the inclusion criteria; 63 of them were included in the study group and 47 in the control group. Mean starting dose was 46 versus 43 mg (p = .25), mean final dose was 52 mg versus 45 mg (p = .03) and dose adjustment was required in 37% versus 11% (p = .002) in the study and control groups, respectively. Twenty-eight percent of anti-Xa measurements in the second trimester were beneath the prophylactic threshold, compared to 11% and 16% in the first and third trimesters, respectively (p = .02). Labors ended with live birth in 91% versus 94% of cases (p = .5), 85% versus 68% of pregnancies were term (p = .05), 11% versus 23% of newborns were low birth weight (p = .1) and placenta-mediated pregnancy complications were documented in 9% versus 19%, (p = .17) in the study group relative to controls, respectively. CONCLUSIONS: The most prominent decrease in anti-Xa levels was observed in the second trimester. Monitored women had significantly more term deliveries and demonstrated a trend toward higher birth weight and fewer placenta-mediated pregnancy complications. Larger studies are needed to confirm improved pregnancy outcome in monitored women.

Augmented Degradation of Factors VIII and IX in the Intermittent Movement State.

The most common clinical presentation of hemophilia A and hemophilia B is bleeding in large joints and striated muscles. It is unclear why bleeding has a predilection to affect joints and muscles. As muscles and joints are involved in intermittent movement, we explored whether this phenomenon could be associated with an impact on factor VIII and IX levels. Purified proteins and a mouse model were assessed using coagulation assays, Western blot analysis and immuno-staining. Movement caused an increase in thrombin activity and a decrease in factor VIII and factor IX activity. The decrease in factor VIII activity was more significant in the presence of thrombin and during movement. Under movement condition, sodium ions appeared to enhance the activity of thrombin that resulted in decreased factor VIII activity. Unlike factor VIII, the reduction in factor IX levels in the movement condition was thrombin-independent. High factor VIII levels were found to protect factor IX from degradation and vice versa. In mice that were in movement, factor VIII and IX levels decreased in the microcirculation of the muscle tissue compared with other tissues and to the muscle tissue at rest. Movement had no effect on von Willebrand factor levels. Movement induces reduction in factor VIII and IX levels. It enables an increase in the binding of sodium ions to thrombin leading to enhanced thrombin activity and augmented degradation of factor VIII. These data suggest a potential mechanism underlying the tendency of hemophilia patients to bleed in muscles and joints.

Extracellular Vesicles of COVID-19 Patients Reflect Inflammation, Thrombogenicity, and Disease Severity.

Severe COVID-19 infections present with cytokine storms, hypercoagulation, and acute respiratory distress syndrome, with extracellular vesicles (EVs) being involved in coagulation and inflammation. This study aimed to determine whether coagulation profiles and EVs reflect COVID-19 disease severity. Thirty-six patients with symptomatic COVID-19 infection with mild/moderate/severe disease (12 in each group) were analyzed. Sixteen healthy individuals served as controls. Coagulation profiles and EV characteristics were tested by nanoparticle tracking analysis (NTA), flow cytometry, and Western blot. While coagulation factors VII, V, VIII, and vWF were comparable, significant differences were found in patients' D-Dimer/fibrinogen/free protein S levels compared to controls. Severe patients' EVs displayed higher percentages of small EVs (<150 nm) with increased expression of exosome marker CD63. Severe patients' EVs displayed high levels of platelet markers (CD41) and coagulation factors (tissue factor activity, endothelial protein C receptor). EVs of patients with moderate/severe disease expressed significantly higher levels of immune cell markers (CD4/CD8/CD14) and contained higher levels of IL-6. We demonstrated that EVs, but not the coagulation profile, may serve as biomarkers for COVID-19 severity. EVs demonstrated elevated levels of immune- and vascular-related markers in patients with moderate/severe disease, and may play a role in disease pathogenesis.

[PERIOPERATIVE MANAGEMENT OF AN ADOLESCENT GIRL WITH SEVERE FACTOR XI DEFICIENCY AND INHIBITORS].

INTRODUCTION: Factor XI (FXI) deficiency is an autosomal bleeding disorder characterized by injury-related hemorrhage, mostly associated with surgical procedures at sites noted for high fibrinolytic activity. Severe FXI deficiency is defined when the FXI level is lower than 15-20 IU/dL. Perioperative prophylactic treatment for high-bleeding-risk surgery in patients with severe FXI deficiency is based on fresh frozen plasma (FFP) transfusions or FXI concentrate (where available). Exposure to FFP and to FXI concentrate may lead to the development of inhibitory antibodies against FXI. This phenomenon occurs mostly in patients with very severe FXI deficiency (baseline FXI <1IU/dL) and is associated with an increased risk of substantial perioperative bleeding, unresponsive to FXI replacement. Thus, in individuals with severe FXI deficiency, routine testing for the presence of inhibitory antibodies against FXI is recommended. We present a 17-year-old adolescent patient with very severe FXI deficiency, who developed an inhibitor to FXI following FFP exposure associated with neurosurgery for medulloblastoma. Prophylactic treatment for subsequent invasive procedures consisted of single low dose (10 mcg/kg) recombinant activated factor VII (rFVIIa) and tranexamic acid (Hexakapron). The procedures were performed uneventfully, with no hemorrhagic or thrombotic complications. In patients with very severe FXI deficiency, the development of inhibitory antibodies following plasma replacement therapy comprises a rare and challenging occurrence. The formulation of a safe and effective evidence-based protocol for hemostatic support in these patients requires multi-center collaboration.

Pregnancy and non-pregnancy related immune thrombotic thrombocytopenic purpura in women of reproductive age.

Pregnancy is a precipitating factor for immune thrombotic thrombocytopenic purpura (iTTP). We compared the clinical course and outcomes of iTTP in women of reproductive age, between those with pregnancy- and non-pregnancy-related iTTP. A review of all reproductive-aged women diagnosed with iTTP during 2010-2019 in seven university hospitals in Israel. Of 42 cases of iTTP, 12 (28.6%) were pregnancy-related. At presentation, the laboratory profiles did not differ significantly between those with pregnancy- and non-pregnancy-related iTTP, including hemoglobin (median 8.4 vs 8.0 g/dL), platelet count (12.5 vs. 11.5 X 109/L); and levels of bilirubin (1.23 vs. 1.82 mg/dL), lactate dehydrogenase (1615 vs. 1701 U/L), creatinine (0.61 vs. 0.79 mg/dL) and anti-ADAMTS13 antibodies titer (75 vs. 82 U/mL). The proportions of women with renal, neurologic, or hepatic involvement were similar between the groups. Cardiac involvement was more common among those with pregnancy-related disease (25.0% vs. 3.3%, P = 0.06). The median number of courses of plasma-exchange therapy was 11 for both groups. All the women were treated with parenteral corticosteroids and the rate of adjunctive treatments did not differ between the groups (P = 0.30). Four women (one-third) with pregnancy-related disease had preeclampsia. Two women (16.7%) with pregnancy-related iTTP died during the acute episode (P = 0.07); no deaths were observed in the non-pregnancy-related group. Among reproductive-aged women with iTTP, most clinical and laboratory profiles were similar between those with pregnancy- and non-pregnancy-related disease. However, the higher rates of cardiac involvement and mortality among women with pregnancy-related iTTP highlight its challenging management.

Circulating heparan sulfate chains and body weight contribute to anti-Xa levels in cancer patients using the prophylactic dose of enoxaparin.

Hospitalized cancer patients are at increased risk of thrombosis and prophylaxis with heparin is recommended. Heparanase is a protein capable of degrading heparan sulfate (HS) chains. The first objective of the study was to examine the effects of weight on anti-Xa levels in cancer patients treated with a fixed dose of enoxaparin as thromboprophylaxis. The second aim was to assess a potential correlation between plasma pre-treatment coagulation parameters and anti-Xa levels in an assumption that heparanase degradation activity towards heparins and HS chains could affect anti-Xa levels. Two blood samples (prior to and 3 h after drug injection) of 76 cancer patients with an indication for prophylaxis with enoxaparin (40 mg) were evaluated for coagulation markers. Sub-prophylactic levels of anti-Xa (< 0.2 U/ml) were found in 16/76 (21%) patients; in 13/76 (13%) patients the values were supra-prophylactic (> 0.5 U/ml). In the subgroup of patients weighing > 80 kg, 7/14 (50%) individuals had a sub-prophylactic level. Overall, anti-Xa levels appeared to correlate with patient's weight (r = - 0.48, p < 0.0001), pre-treatment partial thromboplastin time (PTT), D-dimer, HS, heparanase levels and procoagulant activity. We concluded that plasma anti-Xa levels correlated with patient's weight. A substantial portion of cancer patients receiving enoxaparin prophylaxis was undertreated. For patients > 80 kg, a weight-adjusted prophylactic dose of enoxaparin could be considered. Elevated enoxaparin anti-Xa levels correlated with pre-treatment parameters of coagulation system activation. High pre-treatment HS and elevated plasma anti-Xa levels may potentially serve as biomarkers for the identification of patients at increased thrombosis risk.

[ACQUIRED HEMOPHILIA A AND THE TIMING OF IMMUNOMODULATORY THERAPY].

INTRODUCTION: Acquired hemophilia A is an autoimmune disease affecting men and women equally and is idiopathic in 50% of the cases. As the mortality rate reaches 50%, prompt diagnosis and treatment are needed. Diagnosis is made in a patient with a bleeding manifestation and prolonged PTT (partial thromboplastin time) that is not corrected in a mixing study with normal plasma. The level of antibodies in the plasma is measured by Bethesda units and a level above 5 units is considered high. Patients with a high titer of antibodies are treated with factor VIII, prothrombin complex, recombinant factor VIIa and tranexamic acid, in combination with immunomodulatory therapy, including steroids, cyclophosphamide, rituximab and immunoglobulins. The timing of rituximab therapy remains debatable. To date, it has not been established whether to use it as a first-line or second-line therapy. According to the currently available literature that relies on a database, the use of rituximab as a first-line modality increased survival without increasing the rate of infections, compared to steroids alone or steroids combined with cyclophosphamide. The current article describes a 79-year old woman who presented with diffuse hematomas in the limbs. A rapid diagnosis and treatment, including factor VIII, tranexamic acid, steroids, cyclophosphamide and rituximab as a first-line therapy, facilitated her complete recovery at a one-year follow-up.

[EXPERIENCE IN DIAGNOSTIC ASSAYS FOR HEPARIN-INDUCED THROMBOCYTOPENIA - EXPERIENCE OF A TERTIARY HOSPITAL IN ISRAEL].

AIMS: To analyze the experience of a tertiary medical center in clinical and laboratory diagnosis of suspected HIT. BACKGROUND: The diagnosis of heparin-induced thrombocytopenia (HIT) requires clinical data and laboratory detection of platelet activating factor 4/heparin (PF4/H) antibodies by immunological or functional assays. Although antigen screening assays are widely used, the functional assays are performed only by several expert labs. METHODS: A retrospective review of the Hematology Laboratory database on patients evaluated between the years 2008-2016 at Rambam, identified 412 individuals with clinical suspicion of HIT. Till 2011, 135 cases were screened using particle gel PaGIA (Biorad) and between the years 2012-2016, a total of 277 cases were screened by lateral flow Milenia (Biotec GmbH). All patients diagnosed with HIT were treated with Fondaparinux (Arixtra). Functional assay with heparin/LMWH induced platelet aggregation was performed using light transmission aggregometry (Helena AggRAM) to validate borderline or positive results in indistinct cases. RESULTS: From the tested samples, 63% vs. 75% were negative in PaGIA and Milenia, respectively (P=0.03), and were considered negative for HIT. During 2008-2011, only 38% of cases with non-negative immunoassay results underwent functional aggregation, whereas, in 2012-2016, 83% of such cases were further evaluated. None of the borderline PaGIA samples was positive in the functional assay compared to 13.3% borderline Milenia results; 25% of positive PaGIA and 51.7% of positive Milenia were confirmed by a positive functional HIT assay (P=N.S.). The survival rate among 14 patients with a positive functional assay was 42.7 % (6 patients). CONCLUSIONS: The Milenia assay introduced at our lab in 2012, has improved the screening process. The functional assay provides a more accurate HIT diagnosis. The combined approach of an optimal laboratory and clinical investigation is crucial to obtain a precise HIT diagnosis.

Thrombin is a selective inducer of heparanase release from platelets and granulocytes via protease-activated receptor-1.

Heparanase, known to be involved in angiogenesis and metastasis, was shown to form a complex with tissue factor (TF) and to enhance the generation of factor Xa. Platelets and granulocytes contain abundant amounts of heparanase that may enhance the coagulation system upon discharge. It was the aim of this study to identify the inducer and pathway of heparanase release from these cells. Platelets and granulocytes were purified from pooled normal plasma and were incubated with ATP, ADP, epinephrine, collagen, ristocetin, arachidonic acid, serotonin, LPS and thrombin. Heparanase levels were assessed by ELISA, heparanase procoagulant activity assay and western blot analysis. The effects of selective protease-activated receptor (PAR)-1 and 2 inhibitors and PAR-1 and 4 activators were studied. An in-house synthesised inhibitory peptide to heparanase was used to evaluate platelet heparanase involvement in activation of the coagulation system. Heparanase was released from platelets only by thrombin induction while other inducers exerted no such effect. The heparanase level in a platelet was found to be 40 % higher than in a granulocyte. Heparanase released from platelets or granulocytes increased factor Xa generation by three-fold. PAR-1 activation via ERK intracellular pathway was found to induce heparanase release. In conclusion, heparanase is selectively released from platelets and granulocytes by thrombin interacting with PAR-1. Heparanase derived from platelets and granulocytes is involved in activation of the extrinsic coagulation pathway. The present study implies on a potential anticoagulant effect, in addition to anti-platelet effect, of the new clinically studied PAR-1 inhibitors.

A 14-Year Experience in the Management of Patients with Acquired Immune Thrombotic Thrombocytopenic Purpura in Northern Israel.

Acquired idiopathic thrombotic thrombocytopenic purpura (I-TTP) is a life-threatening microangiopathic disorder usually treated with therapeutic plasma exchange (TPE). The current study assessed the role of rituximab in the treatment of complicated I-TTP. The sequence of TTP events was compared in a group of I-TTP patients treated with TPE and a cohort of refractory or relapsed patients who also received rituximab. This retrospective evaluation included 45 I-TTP patients, treated between January 2000 and October 2013, who underwent at least 3 TPE procedures and were followed up until December 2013 or death. Thirty-one patients with an uncomplicated course received TPE only. Fourteen patients had a complicated course due to either a primary refractory/exacerbated disease (n = 5) or relapse (n = 9) and received rituximab together with TPE. The median number of TPE procedures performed in the first TTP episode in the uncomplicated cohort and groups with primary refractory or relapsed TTP was 11, 27 and 45, respectively. The relapse rates per follow-up year in the uncomplicated I-TTP, primary refractory and relapsed I-TTP groups were 0.18, 0.2 and 0.6 episodes, respectively. After rituximab therapy this rate dropped to 0.2 per year in the relapsed subgroup. In conclusion, about a quarter of patients with I-TTP had a complicated course and experienced a major benefit from rituximab in terms of effectiveness and safety.

Heparanase procoagulant activity, factor Xa, and plasminogen activator inhibitor 1 are increased in shift work female nurses.

Epidemiologic studies indicate on an increased risk of cardiovascular disease and cancer in shift workers, although the underlying mechanism is obscure. Heparanase directly enhances tissue factor (TF) activity leading to increased factor Xa production and subsequent activation of the coagulation system. In the present study, a comparison of coagulation markers among healthy shift working (SW) vs. healthy daytime working (DW) female nurses was performed. Thirty SW and 30 DW female nurses were enrolled. For each of the 60 participants, blood was drawn between 7:00 and 8:00 a.m. and at least 8 h after the last work shift. Plasma was studied for coagulation marker that included TF/heparanase procoagulant activity, TF activity, heparanase procoagulant activity, heparanase level, factor Xa level, plasminogen activator inhibitor 1 (PAI-1), plasminogen, α2-antiplasmin, fibrinogen, global protein C, von Willebrand factor, and D-dimer by chromogenic assays and enzyme-linked immunosorbent assays (ELISAs). Sleep quality was assessed by self-report according to the Pittsburgh Sleep Quality Index. The heparanase procoagulant activity increased by 2-fold and the TF/heparanase procoagulant activity increased by 1.5-fold in SW nurses compared to DW nurses (P < 0.05). Factor Xa levels and PAI-1 levels were significantly higher among SW nurses compared to the DW group (22 vs. 18 ng/ml, P < 0.05, and 32 vs. 22 ng/ml, P < 0.005, respectively). No significant differences were found in the other tested coagulation markers between the study groups. Heparanase procoagulant activity, factor Xa level, and PAI-1 level were significantly higher in SW nurses compared to the DW group. These alterations of blood coagulation activation may potentially contribute to cardiovascular and cancer morbidity.

The coagulation profile of preterm delivery.

INTRODUCTION: Hypercoagulation was suggested to be involved in preterm birth etiology; however, the coagulation state of preterm parturients remains unelucidated. The study aim was to evaluate the haemostatic system of pregnant women with premature uterine contractions (PUC). MATERIALS AND METHODS: The cohort study population consisted of 76 healthy pregnant women admitted with regular PUC. The study group included 38 women who experienced preterm birth; 14 of them had preterm premature rupture of membranes (PPROM). The control group included 38 women who eventually had term delivery. Groups were matched for maternal age, number of births and gestational age at admission. Blood samples were tested for haemostatic parameters and coagulation activation markers. RESULTS: Significantly shorter PT and aPTT were documented in the study compared to control group (25.7±2 vs. 27.4±2.7seconds, P=0.003, and 9.96±0.5 vs. 10.1±0.4seconds, P=0.05, respectively), although differences in absolute values were small. There was no significant difference between the two groups in levels of: fibrinogen, D-dimer, protein C-global, free protein S antigen, factor VIII activity, Von Willebrand factor, plasminogen activator inhibitor-1, prothrombin fragments F1+2 (PT F1+2), tissue factor and tissue factor pathway inhibitor. Women with PPROM had significantly lower PT F1+2 levels compared to those who had preterm delivery with intact membranes (351±99 vs. 561±242pmol/L, P=0.003). CONCLUSIONS: Shortened PT and aPTT, reflecting increased thrombotic activity in maternal plasma, could serve as a marker of real preterm labor in women with premature uterine contractions. Further prospective studies in a larger cohort are warranted to validate these findings.

Neural ectoderm-secreted FGF initiates the expression of Nkx2.5 in cardiac progenitors via a p38 MAPK/CREB pathway.

Vertebrate heart development is derived from paired primordia of anterior dorsolateral mesoderm expressing Nkx2.5 and GATA4 transcription factors. Yet growth factors and intracellular pathways specifying heart precursor gene expression are poorly understood. In the present work, we investigated the signaling events initiating Nkx2.5 expression in Xenopus laevis. We describe here that fibroblast growth factor (FGF) initiates the expression of Nkx2.5 without affecting GATA4. At gastrula, FGF3 is expressed in anterior neural ectoderm, and results presented here indicate that this tissue is involved in the induction of Nkx2.5 expression in neighboring lateral tissues. Further studies indicate that the intracellular p38 MAPK and the CREB transcription factor function downstream of FGF to initiate Nkx2.5 expression. Activation of the p38 MAPK pathway and of the CREB protein is both necessary and sufficient for the initial expression of Nkx2.5. Therefore, we would like to suggest that FGF expressed in anterior neural ectoderm is a major inducer of Nkx2.5 expression in neighboring cells. In these cells, FGF activates an intracellular p38 MAPK signaling pathway and its downstream target, the CREB transcription factor, all participating in the expression of Nkx2.5 in cardiac progenitors.

A p38 MAPK-CREB pathway functions to pattern mesoderm in Xenopus.

Dorsal-ventral patterning is specified by signaling centers secreting antagonizing morphogens that form a signaling gradient. Yet, how morphogen gradient is translated intracellularly into fate decisions remains largely unknown. Here, we report that p38 MAPK and CREB function along the dorsal-ventral axis in mesoderm patterning. We find that the phosphorylated form of CREB (S133) is distributed in a gradient along the dorsal-ventral mesoderm axis and that the p38 MAPK pathway mediates the phosphorylation of CREB. Knockdown of CREB prevents chordin expression and mesoderm dorsalization by the Spemann organizer, whereas ectopic expression of activated CREB-VP16 chimera induces chordin expression and dorsalizes mesoderm. Expression of high levels of p38 activator, MKK6E or CREB-VP16 in embryos converts ventral mesoderm into a dorsal organizing center. p38 MAPK and CREB function downstream of maternal Wnt/beta-catenin and the organizer-specific genes siamois and goosecoid. At low expression levels, MKK6E induces expression of lateral genes without inducing the expression of dorsal genes. Loss of CREB or p38 MAPK activity enables the expansion of the ventral homeobox gene vent1 into the dorsal marginal region, preventing the lateral expression of Xmyf5. Overall, these data indicate that dorsal-ventral mesoderm patterning is regulated by differential p38/CREB activities along the axis.