Hemostatic Challenges Research Articles

Use of Desmopressin in Children With Inherited Platelet Dysfunctions Undergoing Adenotonsillar Procedures

Introduction and goalsAdenotonsillar surgery represents a major haemostatic challenge in paediatric patients with mild inherited platelet dysfunction. While there are recommendations for perioperative haemostatic management, there are no reports of the outcomes with the different recommendations in these children when undergoing adenotonsillectomy. Our objective was to evaluate the management of perioperative bleeding with desmopressin in children with mild platelet dysfunctions who underwent adenotonsillar surgery in our hospital. MethodsWe performed a retrospective study aimed at determining the perioperative bleeding and complication rate in children with mild inherited platelet dysfunction in whom desmopressin was used while undergoing adenotonsillar procedures. ResultsBetween 2004 and 2010, 27 children with mild inherited platelet dysfunction underwent adenotonsillar procedures in our hospital and were treated with desmopressin. One patient developed perioperative bleeding (3.7%) and there was 1 child (3.7%) who presented transitory hypotension as a side effect of desmopressin. ConclusionsThe use of desmopressin allowed adequate perioperative bleeding prophylaxis management in children with mild inherited platelet dysfunction who underwent adenotonsillar procedures without presenting severe complications.

Haemostatic management of extreme challenges to haemostasis in acquired von Willebrand syndrome

Acquired von Willebrand syndrome (AVWS) is a rare hemorrhagic condition for which very little information is available regarding the management of extreme challenges to Haemostasis. The AVWS is more common in the elderly, who are frequently exposed to invasive procedures and/or chemotherapy. Haematopoietic stem cell transplantation (HSCT) is a situation in which the haemostatic capacity is challenged by severe thrombocytopaenia, chemotherapy-associated mucosal barrier breakdown and the need for invasive procedures. In our report, we present and discuss the haemostatic management of a patient with AVWS who was refractory to Von Willebrand factor concentrate replacement during the course of an autologous HSCT to treat multiple myeloma. Patients with AVWS are frequently exposed to high-risk haemostatic challenges, and additional information about the haemostatic management of these situations is necessary.

Neonatal bleeding in haemophilia: a European cohort study

Birth is the first haemostatic challenge for a child with haemophilia. Our aim was to examine the association between perinatal risk factors and major neonatal bleeding in infants with haemophilia. This observational cohort study in 12 European haemophilia treatment centres (HTC) incorporated 508 children with haemophilia A or B, born between 1990 and 2008. Risk factors for bleeding were analysed by univariate analysis. Head bleeds occurred in 18 (3·5%) children within the first 28 d of life, including three intraparenchymal bleeds, one subdural haematoma and 14 cephalohaematomas. Intra-cranial bleeds were associated with long-term neurological sequelae in two (0·4%) cases; no deaths occurred. Assisted delivery (forceps/vacuum) was the only risk factor for neonatal head bleeding [Odds Ratio (OR) 8·84: 95% confidence interval (CI) 3·05-25·61]. Mild haemophilia and maternal awareness of her haemophilia carrier status seemed to be protective (OR 0·24; 95%CI 0·05-1·05 and OR 0·34; 95%CI 0·10-1·21, respectively), but due to the low number of events this was not statistically significant. We found no association between neonatal head bleeding and country, maternal age, parity, gestational age or presence of HTC. Maternal awareness of carrier status protected against assisted delivery (unadjusted OR 0·37; 95%CI 0·15-0·90; adjusted OR 0·47 (95%CI 0·18-1·21).

Uso de desmopresina en niños con disfunción plaquetaria congénita sometidos a adeno y/o amigdalectomía

Introduction and goalsAdenotonsillar surgery represents a major haemostatic challenge in paediatric patients with mild inherited platelet dysfunction. While there are recommendations for perioperative haemostatic management, there are no reports of the outcomes with the different recommendations in these children when undergoing adenotonsillectomy. Our objective was to evaluate the management of perioperative bleeding with desmopressin in children with mild platelet dysfunctions who underwent adenotonsillar surgery in our hospital. MethodsWe performed a retrospective study aimed at determining the perioperative bleeding and complication rate in children with mild inherited platelet dysfunction in whom desmopressin was used while undergoing adenotonsillar procedures. ResultsBetween 2004 and 2010, 27 children with mild inherited platelet dysfunction underwent adenotonsillar procedures in our hospital and were treated with desmopressin. One patient developed perioperative bleeding (3.7%) and there was 1 child (3.7%) who presented transitory hypotension as a side effect of desmopressin. ConclusionsThe use of desmopressin allowed adequate perioperative bleeding prophylaxis management in children with mild inherited platelet dysfunction who underwent adenotonsillar procedures without presenting severe complications.

Hemophilia Gene Therapy

Abstract SCI-48 Over the last decade, five clinical studies on gene therapy for hemophilia A and B using viral and nonviral vectors for liver-restricted or ectopic expression of clotting factor demonstrated safety with no inhibitor formation. Although efficacy in these early phase trials was not achieved because of sustained levels of clotting factor below 1%, these studies form the basis for the second generation of clinical trials. To date, two studies on adeno-associated viral (AAV) vector encoding a FIX transgene for liver-restricted expression are ongoing. In one study using a modified AAV genome pseudotyped with serotype 8, emerging data are encouraging and sustained therapeutic levels of FIX have been obtained in a dose-dependent manner. Novel approaches for hemophilia have been explored targeting hematopoietic stem cells (HSC) using lentiviral vectors for expressing FVIII or FIX genes. Transgene expression under the control of either a non-lineage specific promoter or a platelet-specific promoter showed biological activity and improvement of the disease phenotype. The platelet-restricted approaches did not increase circulating plasma clotting factor levels, but they resulted in enrichment of the factor at the injury site upon platelet activation. An advantage of targeting FVIII expression and storage in platelets is the protection of FVIII from neutralizing antibodies (inhibitors). In this model, platelet-FVIII provided superior hemostasis than elevated plasma FVIII levels upon hemostatic challenges in the microcirculation and macrocirculation. Recently, platelet-restricted expression of human FVIII gene using a lentiviral vector for ex vivo transduction of canine HSC resulted in improvement of the disease phenotype in severe hemophilia A dogs without unwanted immune responses to the transgene. Additional strategies to optimize gene- and/or cell-based approaches have also focused on use of clotting factors with enhanced biological activities will be discussed. Together with the generation of novel vectors, further enhancement of both the efficacy the safety of these approaches is envisioned. Disclosures: Arruda: Pfizer: Research Funding.

Correlation of a Condensed Bleeding Score with New Diagnosis of a Congenital Bleeding Disorder in Patients Referred to a Tertiary Centre

Abstract 1226▪▪This icon denotes a clinically relevant abstractThe performance and utility of a condensed bleeding score (Bowman et al, J Thromb Haemost., 2008;6:2062) in relation to the diagnosis of a congenital bleeding disorder in new referrals to a regional haemostasis clinic over an 8 month period is presented. Between November 2010 and June 2011, 50 patients over the age of 16 (median age, 31 years; range, 16–79), including 32 females, were referred for investigation of a possible congenital bleeding disorder following detection of abnormal coagulation results and/or presentation with a bleeding history. A bleeding score was performed as part of their initial assessment. 12(24%) patients were from local referral and 38(76%) patients were referred from other hospitals in the region for further investigation of a suspected bleeding disorder. Basic coagulation tests (activated partial thromboplastin time (APTT), prothrombin time Clauss fibrinogen and platelet count) were normal in the referred patients from other centres. 50% (6/12) of the local referrals were for investigation of a prolonged APTT detected on routine coagulation screening prior to major surgery.The median bleeding score was 6 with a range of −1 to 14 (Table 1). The presence of a congenital bleeding disorder was confirmed in 31 of the 50 patients (62%), including 19/31 (61%) of the female patients and 12/31(39%) of the males. Correlation of an abnormal bleeding score (score ≥ 4) with diagnosis of a congenital bleeding disorder was only seen for diagnosis of type 1 Von Willebrand Disease (VWD) (Table 2). Analysis of the cases with low scores and abnormal results identified two groups of patients; firstly, those who had not yet had a significant haemostatic challenge, and secondly, those in whom the abnormal coagulation results were explained by a non-haemostatically significant reduction in a coagulation factor level (e.g. FVII, 15%; dysfibrinogenaemia; F XII deficiency). These clinically insignificant laboratory abnormalities explain the discrepancy between the number of patients with abnormal laboratory tests (35) and the number of patients diagnosed with a congenital bleeding disorder (31).Table 1Bleeding score (range)Number of patients with normal lab resultsNumber of patients with abnormal lab results−1 to +1382–44105–74128–102311–1422Total1535Table 2DiagnosisNumber of patientsMedian bleeding scoreAge rangeType 1 VWD116 (4–10)17–51Type 2 VWD48 (5–13)17–36Factor XI123 (1–8)17–76Platelet function defect46 (2–9)17–57Compared to previous reports the range of scores found with this assessment tool was narrow and could not exclude patients from further laboratory assessment. However the condensed bleeding score has only been validated prospectively for the diagnosis of type 1 VWD and all patients in this cohort who were diagnosed with type 1 VWD had an abnormal bleeding score (≥ 4). This observation supports the role of this scoring system in the assessment of patients for type 1 VWD. The use of the condensed bleeding score in assessing patients with suspected factor XI deficiency is difficult due to the lack of a phenotypic relationship between residual factor XI activity and a bleeding tendency. Furthermore, although factor XI deficiency is a rare congenital bleeding disorder in our cohort of patients 12/31(39%) were diagnosed with factor XI deficiency. This may explain the overall lack of correlation between bleeding score and diagnosis of a congenital bleeding disorder. Patients who have an abnormal bleeding score but normal laboratory tests need consideration of further investigations before concluding they are normal. The possibility of an acquired bleeding disorder should be considered. A thorough drug history is also important as one of the patients with a bleeding score of 14 was taking a non-steroidal anti-inflammatory drug.The use of the condensed bleeding score in the detection of congenital bleeding disorders other than type 1 VWD requires further validation in a larger number of patients. Disclosures:No relevant conflicts of interest to declare.

ARFI Ultrasound Monitoring of Hemorrhage and Hemostasis In Vivo in Canine Von Willebrand Disease and Hemophilia

A validated method for assessing hemostasis in vivo is critical for testing the hemostatic efficacy of therapeutic agents designed for patients with bleeding disorders such as von Willebrand disease (VWD) and hemophilia A. We hypothesize that rate of bleeding and time to hemostasis can be monitored in vivo by acoustic radiation force impulse (ARFI) ultrasound. We performed ARFI imaging following 12-gauge needle puncture of hind limb muscle encompassing an ∼2 mm vein in six normal, eight naïve hemophilia A before and after infusing canine factor VIII, three hemophilia A expressing canine factor VIIa following gene transfer, and two naïve VWD dogs. Serial data sets were processed with custom software to (1) estimate the rate of hemorrhage and (2) estimate the time of hemostasis onset. The rate of hemorrhage during the first 30 min following puncture was markedly increased in the VWD dogs relative to normal but was not significantly different between normal, naïve hemophilia A or hemophilia A expressing cFVIIa. ARFI-derived times to hemostasis were significantly longer in naïve hemophilia A dogs than in normal dogs and were shortened by canine coagulation factors VIII and VIIa. These data support our hypothesis that rate of hemorrhage and time to hemostasis in vivo in response to a standardized hemostatic challenge can be detected by ARFI ultrasound in canine models of VWD and hemophilia. These data also suggest that the ARFI-monitored hemostatic challenge is relevant for in vivo testing of the hemostatic efficacy of therapeutic clotting factor replacement products used to treat inherited bleeding disorders.

Management of pregnancy, labour and delivery in women with inherited bleeding disorders

Pregnancy, labour and delivery present intrinsic haemostatic challenges to women with and carriers of bleeding disorders and their offspring. Deficiency of fibrinogen and factor XIII are associated with miscarriage, placental abruption and foetal loss. The risk of antenatal complications including antepartum haemorrhage is unknown in women with other bleeding disorders. There is a significant risk of postpartum haemorrhage (primary and secondary) in women with all types of bleeding disorders. This can be serious and life threatening in those with severe defects such as Bernard Soulier syndrome and Glanzmann's thrombasthenia. Three to four percent of infants with haemophilia experience cranial bleeding that occurs during labour and delivery. The safest method of delivery for affected babies remains controversial. However, the rate of planned Caesarean section is increasing among known carriers of haemophilia. If vaginal delivery is planned, prolonged labour and difficult delivery especially vacuum extraction are associated with the highest risk of cranial bleeding and should be avoided. The optimal management of pregnancy in women with inherited bleeding disorders requires a multidisciplinary approach and advanced individualized management plan taking into consideration obstetric and bleeding risk factors. Women with mild or moderate bleeding disorders can be managed at their local maternity unit in close collaboration with a tertiary centre. However, those with severe or rare disorders or carrying an affected infant should be managed in a tertiary centre with an onsite Haemophilia centre.

Inherited and acquired factor V deficiency

The clotting factor V, also known as proaccelerin or labile factor, is synthesized by the liver and possibly by the megakaryocytes. Factor V exerts a pivotal role in hemostasis, as it participates in both procoagulant and anticoagulant pathways, being an essential cofactor of the prothrombinase complex in the former case and participating in the inactivation of factor VIII (FVIII) in the latter. Isolated factor V deficiency due to mutations in the F5 gene is a rare inherited coagulopathy typically associated with a broad spectrum of bleeding symptoms, ranging from easy bruising, delayed bleeding after haemostatic challenges such as trauma or surgery to more severe joint bleeds. The combined deficiency of factor V and FVIII, commonly known as F5F8D, is a recessive disorder not attributable to the association of isolated factor V and FVIII deficiencies, but rather to defective intracellular processing of both proteins due to mutations involving the LMAN1 and MCFD2 genes, which encode two proteins forming an essential cargo receptor complex. Overall, patients affected by F5F8D do not bleed more in terms of both frequency and severity than those carrying specific deficiencies of both factors and the bleeding phenotype is generally mild. Although now increasingly rare, inhibitors directed against factor V may also develop in individuals of any age and are characterized by a very heterogeneous clinical phenotype. The aim of the current review is to provide an overview on the physiopathology, diagnostics, and clinical management of both inherited and acquired factor V deficiency.

Elevated factor VIII enhances thrombin generation in the presence of factor VIII-deficiency, factor XI-deficiency or fondaparinux

Background Increased levels of factor VIII occur as a response to vascular injury and/or inflammation, and may increase thrombotic risks. In contrast, factor VIII deficiency poses a major hemostatic challenge. The role of factor VIII in modulating hemostasis/thrombosis was investigated in plasma models of hypocoagulable and hypercoagulable state using thrombin generation (TG) assay. Methods TG was performed in undiluted/diluted control, FVIII-deficient, FVIII-deficient with low antithrombin (AT activity, ~ 59%), and factor XI-deficient plasma samples using relipidated tissue factor (TF, 2 pM) or dilute Actin as activators. The impact of elevated FVIII on TG was simulated by adding Humate-P (0 to 3 U/ml) to the above plasma samples. In fondaparinux (1 μg/ml) treated plasma with normal or lower AT activity effects of Humate-P vs. 60 nM of recombinant activated factor VII (rFVIIa) were also evaluated. Results Humate-P increased TG concentration dependently in undiluted and diluted control plasma with TF activation. With Actin activation, only the concentration dependent shortening of lag time, but no change in peak thrombin was observed. In FVIII-deficient, FVIII-deficient with low AT, and FXI-deficient samples, 3 U/ml of Humate-P increased TG, and decreased its onset with either activator. The reduced peak thrombin due to fondaparinux was reversed with Humate-P (3 U/ml) more than with rFVIIa. Elevated FVIII levels seem to favor intrinsic tenase formation and antagonize fondaparinux because anti-FIXa aptamer added to fondaparinux effectively attenuated TG. Conclusion Elevated FVIII supports the propagation of TG via intrinsic tenase formation under low TF condition, factor XI deficiency or in the presence of fondaparinux.

Hemostatic Challenges In Patients Treated with Eltrombopag

AbstractAbstract 2511 Introduction:Chronic immune thrombocytopenia (ITP) is an autoimmune disease in which antiplatelet-antibodies induce platelet destruction and impair platelet production, resulting in chronically low platelet counts. Patients with chronic ITP may require invasive procedures associated with bleeding (hemostatic challenges) that cannot be undertaken if the platelet count is unacceptably low. Eltrombopag is an oral, nonpeptide, thrombopoietin receptor (TPO-R) agonist developed to increase platelet counts in various conditions associated with thrombocytopenia; its use may facilitate undertaking invasive procedures in patients with chronic ITP, reducing the need for additional supportive requirements including cellular blood products. Methods:Across the eltrombopag ITP clinical trials, information about hemostatic challenges was collected retrospectively (TRA100773A and B) and prospectively (REPEAT, RAISE, EXTEND). Basic demographic information, platelet counts before and after the procedures, type of procedure, need for additional treatment to increase platelet counts (one week before and after the intervention), use of blood products, and where possible, assessment of bleeding and bleeding complications were recorded. For the purpose of this analysis, minor invasive procedures (eg, dental cleaning, endoscopy, bone marrow biopsy) were distinguished from major invasive procedures (splenectomy, laparotomy, hip replacement, aortic aneurysm repair, arthroplasty). Results:Seventy-seven patients underwent 120 invasive procedures while enrolled in clinical trials with eltrombopag. The median age of patients undergoing invasive procedures was 54 years; the median duration of treatment at the time of all procedures was 131 days. 112 invasive procedures were performed in patients while receiving eltrombopag, compared to 8 procedures among patients while receiving placebo. 65 (54%) were considered to be major and 55 (46%) were considered to be minor. The median platelet count closest to minor or major invasive procedures in patients receiving eltrombopag was higher than in those receiving placebo (Table). For minor procedures, rescue ITP medication was required in 9/52 (17%) procedures in patients treated with eltrombopag and in 1/3 (33%) procedures in patients receiving placebo. For major procedures, rescue ITP medication was required in 14/60 (23%) procedures in patients treated with eltrombopag and 3/5 (60%) procedures in patients receiving placebo. One bleeding complication was reported in an eltrombopag-treated patient with colon cancer who, on the first post-operative day after a colectomy, experienced a pulmonary embolism requiring anticoagulation and had an intra-abdominal hemorrhage on post-operative day 2. Conclusions:No difference in use of periprocedural blood products between groups was discernable, possibly due to the low frequency of bleeding events reported. Although the number of patients who did not undergo procedures due to thrombocytopenia was not captured, data from 77 patients undergoing 120 invasive procedures suggest that by achieving a sustained platelet increase in patients with chronic ITP, eltrombopag facilitates the undertaking of medical and surgical procedures associated with bleeding.TableInvasive ProceduresMedian Proximal Platelet Count, Gi/LMinor, nBeforeAfterEltrombopag, 528277Placebo, 32022Major, nEltrombopag, 609281Placebo, 51726 Disclosures:Tarantino: GlaxoSmithKline, Novo Nordisk, Talecris, Baxter, Cangene: Honoraria, Research Funding, Speakers Bureau. Fogarty: GlaxoSmithKline: Honoraria, Research Funding. Mayer: GlaxoSmithKline: Employment, Equity Ownership. Vasey: GlaxoSmithKline: Employment. Brainsky: GlaxoSmithKline: Employment.

Evaluation of the Severity and Pattern of Epistaxis In Children with a Mucocutaneous Bleeding Disorder and In Healthy Children Using a Standardized Questionnaire

AbstractAbstract 713Epistaxis is a common symptom in children with mucocutaneous bleeding disorders such as von Willebrand disease (VWD) or a platelet function disorder (PFD), but also occurs in healthy children. Determining the features of epistaxis that suggest underlying hemostatic defects would help distinguish which children with epistaxis should undergo laboratory testing to screen for an inherited bleeding disorder. This is particularly significant in young children who may not yet have been exposed to a hemostatic challenge. However, there is no standardized approach to determine which children presenting with epistaxis should undergo testing. The aim of this study was to describe the severity and pattern of epistaxis in children with VWD or a PFD vs. healthy children using the PBQ, a standardized, validated Pediatric Bleeding Questionnaire (Bowman et al, J Thromb Haemost 2009:7;1418). PBQ epistaxis scores were assigned depending on clinical severity of the most severe episode (0: no epistaxis or trivial, ≤5/yr; 1: >5/yr or >10 min duration; 2: consultation with a healthcare professional; 3: packing, cauterization or antifibrinolytics; 4: blood transfusion, replacement therapy or desmopressin); scores of ≥2 were considered clinically significant. Epistaxis scores and features (frequency, duration, onset (spontaneous or not), site (uni- or bilateral), presence of seasonal correlation, whether cessation required medical intervention) were retrospectively reviewed in 107 questionnaires administered to patients/parents of patients with a known diagnosis of VWD (type 1, n=60 [definite, with abnormal laboratory results at least twice + a significant bleeding history, n=37; possible, with abnormal laboratory results at least once ± a significant bleeding history, n=23]; type 2, n=8; type 3, n=16) or a PFD (Glanzmann thrombasthenia, dense granule disorder, MYH9-related disorder, Noonan syndrome, Ehlers-Danlos syndrome, unspecified; n=23) and in 163 questionnaires administered to healthy children (Bowman et al, J Thromb Haemost 2009:7;1418; Biss et al, J Thromb Haemost 2010:8;950; Biss et al, J Thromb Haemost 2010:8;1416). Epistaxis was present in 66/107 patients (62%) (definite type 1 VWD: 62%; possible type 1 VWD: 48%; type 2 VWD: 38%; type 3 VWD: 75%; PFD: 74%) with a median age of 12 yrs (range: 0.6–18.3 yrs), and in 56/163 healthy children (34%), with a median age of 11.5 yrs (range: 1.5–17.0 yrs). 59% of the 66 patients with epistaxis were males, as were 50% of the 56 controls with epistaxis. The mean PBQ epistaxis score in these 66 patients with VWD or a PFD was greater, 2.1 (range: 0–4), than in the 56 controls, 0.4 (range: 0–2) (P<0.0001). The mean PBQ epistaxis score in patients varied according to underlying diagnosis and was greater in those with type 3 VWD (3.1) than in the patients with definite type 1 VWD (2.2), possible type 1 VWD (0.8), type 2 VWD (2.0) or a PFD (2.1) (P<0.05). There was no effect of age or gender on PBQ epistaxis scores within patient and control groups. 38/66 patients (58%) had a clinically significant score compared to only 5/56 (9%) of controls (P<0.0001). Epistaxis frequency (>5 episodes/yr) was greater in patients than controls, 44/66 (67%) vs. 11/56 (20%) (P<0.0001). More patients (41/66; 62%) had epistaxis lasting >10 min compared with controls (5/56; 9%) (P<0.0001). Most patients and controls reported spontaneous onset of epistaxis − 57/66 (86%) and 44/56 (79%), respectively (P=0.2316). However, a greater number of patients bled from both nostrils (51/66; 77%) than controls (15/56; 27%) (P<0.0001). Seasonal correlation of epistaxis occurred less frequently in the patient group; 14/66 patients (21%) had epistaxis only during 1 or 2 specified seasons of the year while 24/56 controls (43%) had seasonal correlation of epistaxis (P=0.0219). More patients (38/66; 58%) required medical intervention for cessation of epistaxis than controls (5/56; 9%) (P<0.0001). In conclusion, the standardized PBQ is useful in the assessment of epistaxis pattern and severity in children with a mucocutaneous bleeding disorder – VWD or a PFD. The severity score is significantly greater in patients vs. controls. Features of epistaxis suggestive of an underlying bleeding disorder include: epistaxis frequency >5 episodes/yr; duration >10 min; epistaxis occurring from both nostrils; lack of seasonal correlation; and need for medical intervention for cessation. Disclosures:No relevant conflicts of interest to declare.

Platelet Disorders in Adolescents

Acquired and inherited platelet disorders can present with bleeding symptoms during adolescence. Platelet disorders include disorders of platelet number and disorders of platelet function. In adolescent females with platelet disorders, menorrhagia is a common presenting bleeding symptom. Other associated bleeding symptoms are also primarily mucocutaneous, including epistaxis and bruising. Excessive bleeding may also occur after hemostatic challenges. Diagnosis of disorders of platelet function usually requires light transmission platelet aggregometry. Management of menorrhagia in the adolescent with platelet disorders requires both hematologic and gynecologic treatment.

ISTH/SSC bleeding assessment tool: a standardized questionnaire and a proposal for a new bleeding score for inherited bleeding disorders.

ISTH/SSC bleeding assessment tool: a standardized questionnaire and a proposal for a new bleeding score for inherited bleeding disorders.

Postpartum changes in the level of factor VIII and von Willebrand factor

AimTo determine the exact time period when clotting factor levels decrease post partum.BackgroundPregnancy is a haemostatic challenge associated with elevated levels of clotting factors. However it is not known when...

Multidisciplinary Approach to the Challenge of Hemostasis

A multidisciplinary panel consisting of experts chosen by the 2 chairs of the group representing experts in anesthesiology, blood banking, hematology, critical care medicine, and various surgical disciplines (trauma, cardiac, pediatric, neurologic, obstetrics, and vascular) convened in January 2008 to discuss hemostasis and management of the bleeding patient across different clinical settings, with a focus on perioperative considerations. Although there are many ways to define hemostasis, one clinical definition would be control of bleeding without the occurrence of pathologic thrombotic events (i.e., when balance among procoagulant, anticoagulant, fibrinolytic, and antifibrinolytic activities is achieved). There are common hemostatic challenges that include lack of scientific evidence and standardized guidelines for the use of therapeutic drugs, need for reliable and rapid laboratory tools for measuring hemostasis, and individual variability. Clinically meaningful and accurate real-time laboratory data reflecting a patient's hemostatic status are needed to guide treatment decisions. Current available routine laboratory tests of hemostasis (e.g., platelet count, prothrombin time/international normalized ratio, and activated partial thromboplastin time) do not reflect the complexity of in vivo hemostasis and can mislead the clinician. Although point-of-care coagulation monitoring tests including measures of thromboelastography/elastometry provide insight into overall hemostatic status, they are time-consuming to perform, complex to interpret, and require trained personnel. There is a particular need to develop laboratory tests that can measure the effects of anticoagulant and antiplatelet agents for individual patients, predict bleeding complications, and guide therapy when and if treatment with blood products or pharmacologic drugs is required. Formation of an organization comprised of specialists who treat bleeding patients will foster multidisciplinary collaborations and promote discussions of the current state of hemostasis treatment and future priorities for hemostasis research. Controlled trials with clinically meaningful end points and suitable study populations, as well as observational studies, investigator-initiated studies, and large registry and database studies are essential to answer questions in hemostasis. Because of the complexities of maintaining hemostatic balance, advances in hemostasis research and continuing communication across specialties are required to improve patient care and outcomes.

Efficacy and Safety of Continuous Expression of An Improved Variant of FVIIa On Murine Hemostasis with or without Inhibitors to Human Factor IX.

Abstract 2466Poster Board II-443Recombinant human activated Factor VII (rhFVIIa) is extensively used in the management of hemophilic inhibitor patients. As an alternative, using an adeno-associated viral (AAV) vector as a delivery vehicle for an engineered transgene for activated canine FVII (cFVIIa), our large animal studies in hemophilia A and B dogs demonstrated effective, long-term hemostasis and elimination of the bleeding episodes. However, the relatively high vector doses required for efficacy underscores the need for improvements in the delivery vector and/or the transgene itself. To address this, we decided to investigate whether changes in the catalytic domain of FVIIa can improve its coagulant activity, thus resulting in lowering of the effective vector dose. Using the murine version of our engineered FVIIa (mFVIIa) as a backbone, we generated a variant with four amino-acid substitutions in the catalytic domain of murine FVIIa (L305V/A314E/K337A/I374Y, mFVIIa-VEAY), based on an existing enhanced activity human FVIIa variant. As we have previously described (Amer. Soc. Hematol. Meeting 2006, #3276; Amer. Soc. Gene Cell Ther. Meeting 2008, #127), purified mFVIIa-VEAY exhibited 6-7 fold higher intrinsic coagulant activity than mFVIIa. The hemostatic properties relative to mFVIIa were determined following AAV-mediated gene delivery in hemophilia A (HA) mice. Administration of AAV-mFVIIa-VEAY at 40-100 fold lower vector dose (1.2 - 3E10 vector genomes [vg]/mouse) than AAV-mFVIIa (1.2E12 vg/mouse, High dose) resulted in ∼10-fold lower expression of the mFVIIa-VEAY (based on RNA transcript levels), but was sufficient to result in long-term (>12 weeks) normalization of the hemophilic activated partial thromboplastin time (aPTT), similar to that seen with high dose AAV-mFVIIa (P>0.05). In addition, it improved hemostasis similar to that seen with AAV-mFVIIa following in vivo hemostatic challenges (tail clip assay and ferric chloride carotid artery injury). As an extension of these studies, we have now investigated the ability of low-dose mFVIIa-VEAY gene delivery to improve hemostasis in the presence of inhibitory antibodies to human Factor IX (hFIX). Using an adjuvant, we developed a protocol for generating persistent (>3 months, ongoing) inhibitory antibodies to human Factor IX (hFIX) in hemophilia B (HB) mice. Using the low dose of 3E10 vg/mouse, we administered AAV-mFVIIa-VEAY in HB mice with high titer (7-30 Bethesda units [BU]) to hFIX. In agreement with our previous results, following gene transfer, we observed normalization of the hemophilic aPTT at the expression plateau (4 weeks [ongoing]; P>0.05 vs. wildtype mice; P<0.05 vs. untreated HA mice with similar titers of inhibitory antibodies to hFIX). However, despite the clear efficacy observed in hemophilia mice following low-dose mFVIIa-VEAY gene delivery, our previous observations with mFVIIa-VEAY overexpression (following a 1.2E12 vg/mouse of administered AAV [High dose]) in HA mice indicated a 70% reduction in survival within 6 weeks post vector administration. This coincided with a time-dependent increase in plasma thrombin-antithrombin levels that peaked at 4 weeks post AAV infusion (∼70 ng/ml) and was not observed in untreated HA mice (∼ 20ng/ml, P>0.05), HA mice treated with low-dose mFVIIa-VEAY (∼40ng/ml, P>0.05) or HA mice treated with AAV-mFVIIa (∼35ng/ml, P<0.05). Further extending these studies, histological examination from organs of deceased mice revealed thrombi in the heart as well as gross loss of lung structure. Immunofluoresence microscopy demostrated fibrin deposition in the lung parenchyma, suggesting a compromise of the lung vascular bed in the deceased mice. The identical experiment using hemostatically normal mice resulted in 100% mortality within 6 weeks with similar histological findings compared to HA mice following high dose AAV-mFVIIa-VEAY administration. Overall, our results using a high activity mFVIIa variant, demonstrate similar efficacy to mFVIIa but at a substantially reduced vector dose/expression, in a gene transfer setting for hemophilia with or without inhibitors. This variant thus serves as a potential candidate that can lower the effective vector dose in FVIIa gene-based studies in large animal models of hemophilia. However, the increased mortality observed in mice expressing high levels of mFVIIa-VEAY, warrants further investigation into the long-term safety of coagulation proteases with enhanced activity. Disclosures:High:Novo Nordisk: Grant Review Panel.

The Development of Novel Hemostatic Bypassing Molecules.

Abstract SCI-20Blood coagulation factors VIII and IX are part of the intrinsic pathway and play a critical role in maintaining normal hemostasis by activating factor X. Deficiency of either of these proteins caused by mutations in the genes encoding FVIII or FIX leads to hemophilia A or B (HA and HB), respectively. In each disease, there is impairment of the intrinsic pathway with inadequate thrombin (IIa) generation and defective hemostasis. Unfortunately, ∼20-30% of patients with FVIII deficiency and ∼3-5% of patients with FIX deficiency develop inhibitory antibodies to infused factor replacement products. This has prompted the research community to develop so-called “bypass strategies” which use other coagulation factors to provide hemostasis in these patients. In principle, infusion of FXa should bypass deficiencies in the intrinsic pathway; however it is generally thought that it has limited utility since the infused FXa could cause excessive activation of coagulation and/or more importantly, FXa is rapidly inactivated by plasma inhibitors resulting in a very short half-life (<2 min). We recently characterized variants of FXa (FXa-I16L and FXa-V17a) which have “zymogen-like” properties that could circumvent these associated problems (JBC 2008; 283: 18627). For example we have found that i) these proteins have an incompletely formed active site, making them resistant to plasma protease inhibitors; ii) in the absence of FVa, the FXa variants are, in general, refractory to active site functions and thus do not activate FVII, FV, FVIII, and prothrombin very well; and iii) the variants are thermodynamically rescued by FVa; thus at the site of injury on the activated platelet surface, where FVa is present, prothrombinase rapidly forms generating a burst of thrombin. We have begun to exploit these unique properties and evaluate whether these FXa variants could be effective and safe in bypassing the hemophilic phenotype both in vitro and in vivo. Clotting and IIa generation assays in human HA, HB and inhibitor plasma revealed that FXa-I16L could completely restore IIa generation in a FVa-dependent fashion. Furthermore, the zymogen-like conformation protects FXa-I16L in human plasma as it has a prolonged half-life (∼2 hr) versus wt-FXa (<2 min). In vivo studies using HB mice revealed that administration of FXa-I16L via tail vein almost completely corrected the prolonged aPTT. The aPTT was shortened for more than 2 hours and returned close to the starting value after 24 hr. In these experiments, infusion of the protein was well tolerated as platelet levels were unaffected over the course of the experiment with little or no change in the values for TAT, D-dimer, and fibrinogen. Next we tested whether the improvement of the clotting times was associated with in vivo hemostatic performance. Using three separate injury models (tail clip assay, FeCl2 carotid artery injury model, and cremaster muscle laser injury model) infusion FXa-I16L provided effective hemostasis. This was in contrast to the infusion of wild-type FXa which was ineffective. Taken together our data show that FXa-I16L is highly effective in correcting the hemostatic defect in human hemophilic plasma. Furthermore it improves the hemophilic phenotype in HB mice following a series of hemostatic challenges and can restore thrombus formation upon injury at both micro and macrocirculation levels. Thus zymogen-like variants of FXa have properties that indicate their ability to serve as superior therapeutic procoagulants for bypassing deficiencies upstream of the common pathway. DisclosuresCamire:Wyeth: Patents & Royalties, Research Funding. Bunce:Wyeth: Research Funding. Ivanciu:Wyeth: Research Funding. Downey:Wyeth: Research Funding.

Evaluation of Bleeding Symptoms in Children with An Inherited Mucocutaneous Bleeding Disorder.

Abstract 1292Poster Board I-314 IntroductionAccurate assessment of bleeding symptoms is an essential step in the diagnosis of an inherited mucocutaneous bleeding disorder (IMBD). This is difficult in childhood when symptoms such as bruising and epistaxis are frequently noted in normal children. In addition, children may not have been exposed to hemostatic challenges such assurgery, dental extraction and menarche. Knowledge of the spectrum of presenting features of an IMBD may aid diagnosis. The MCMDM-1 VWD Bleeding Questionnaire, which uses a symptom-specific grade of -1 to 4 (Tosetto et al., J Thromb Haemost 2006:4;766), was recently modified for use in a pediatric population by inclusion of questions related to pediatric-specific bleeding symptoms. The Pediatric Bleeding Questionnaire (PBQ) has been validated in the assessment of bleeding symptoms in children and a score of 2 or greater predicts a diagnosis of VWD (Bowman et al., J Thromb Haemost 2009; doi:10.1111/j.1538-7836.2009.03499.x). This present study applied the PBQ to a cohort of children with a prior diagnosis of an IMBD in order to evaluate the prevalence and severity of specific bleeding symptoms. Patients and MethodsThe PBQ was used to determine bleeding score for 123 children with a prior diagnosis of VWD or a platelet function disorder (PFD) at The Hospital for Sick Children, Toronto, or Kingston General Hospital, Kingston, by interview of parents/children. A clinically-significant bleeding symptom was defined as one severe enough to require consultation with a healthcare professional, medical/surgical intervention or blood transfusion. Results100 children had a diagnosis of VWD and 23, a PFD (Glanzmann thrombasthenia: 4; dense granule deficiency: 9; MYH9-related disease: 3; Noonan syndrome: 2; Ehlers-Danlos syndrome: 2; undefined PFD: 3). 65 were male and 58 female with a median age of 10.7 years (range: 0.6-18). Mean bleeding score was 8.3 for the entire cohort (range: 0-29) and varied between diagnostic groups (definite type 1 VWD: 9.5 (range: 2-18); possible type 1 VWD: 3.0 (range: 0-15); type 2 VWD: 11.8 (range: 3-17); type 3 VWD: 14.6 (range: 4-29); PFD: 9.5 (range: 1-20). The prevalence of each of the bleeding symptoms is shown in Table 1. The most frequent clinically-significant bleeding symptoms in the cohort were: menorrhagia, 57% (of menstruating females); epistaxis, 36%; prolonged bleeding from minor wounds, 35%; and bleeding after tooth extraction, 30%. The most frequent clinically-significant bleeding symptoms did not vary greatly between each of the diagnostic groups but oral cavity bleeding, hemarthrosis, gastrointestinal bleeding and hematoma were more frequent in children with type 3 VWD. All symptoms were less frequent in children with possible type 1 VWD. Menorrhagia was a prevalent symptom in menstruating females in all diagnostic groups. Clinically-significant pediatric-specific bleeding symptoms were seen in 20% of children. The most frequent were: post-circumcision bleeding, 35% (of circumcised males); cephalohematoma, 7%; macroscopic hematuria, 4%; and umbilical stump bleeding, 3%. These symptoms occurred most frequently in children with type 3 VWD, type 2 VWD or PFD. ConclusionsChildren with an IMBD frequently report menorrhagia, epistaxis, prolonged bleeding from minor wounds and bleeding after tooth extraction. Pediatric-specific bleeding symptoms also occur. There are differences in prevalence of symptoms between subtypes of VWD and PFDs. This information can assist the evaluation of children with bleeding symptoms.Table 1Bleeding symptom*% of children with a clinically-significant scoreDefinite type 1 VWD (n=40)Possible type 1 VWD (n=38)Type 2 VWD (n=6)Type 3 VWD (n=16)Platelet function disorder (n=23)All (n=123)Epistaxis48850564336Cutaneous351317253025Minor wounds481350384335Oral cavity18333502219Tooth extraction401133383930Gastrointestinal13017311311Surgery23550383523Muscle hematoma135025410Hemarthrosis353344911Central nervous system330602Pediatric-specific**18533383520*Menorrhagia (expressed as a percentage of menstruating females): Definite type 1 VWD, 46% (6/13); Possible type 1 VWD, 33% (1/3); Type 2 VWD, 50% (1/2); Type 3 VWD, 100% (2/2); PFD, 100% (3/3); All, 57% (13/23).**Post-circumcision bleeding, cephalohematoma, macroscopic hematuria, umbilical stump bleeding, conjunctival hemorrhage, post-venepuncture bleeding DisclosuresNo relevant conflicts of interest to declare.

Advanced twin abdominal pregnancy: diagnostic and therapeutic challenges

Abdominal pregnancy remains a diagnostic challenge despite the remarkable improvements and widespread availability of antenatal ultrasound. A case of advanced twin abdominal pregnancy that was diagnosed after failed induction of labor is presented highlighting the diagnostic and hemostatic challenges encountered. The role and limitations of antenatal ultrasound as well as the importance of adhering to basic ultrasound imaging principles are emphasized as a way of ensuring an early diagnosis and minimizing the devastating consequences associated with this rare variety of ectopic pregnancy.