Increased Bleeding Tendency Research Articles

Severe Upper Gastrointestinal Bleeding in Heartmate II Induced by Acquired von Willebrand Deficiency: Anticoagulation Management

Patients treated with continuous flow assist devices may have increased bleeding tendencies due to an induced high molecular weight von Willebrand factor (VWF) multimer deficiency. We report a patient supported with a HeartMate II (Thoratec, Pleasanton, CA) who developed severe gastrointestinal bleeding refractory to conventional therapy and needing a total of 60 transfusions. After documenting the lack of large VWF multimers, suggestive of a defective platelet function, the patient was switched from aspirin to warfarin therapy (target international normalized ratio between 1.5 and 2.0). Three days after changing the anticoagulant regimen, the patient stopped bleeding and required no further transfusion.

Inhibitor incidence after intensive FVIII replacement for surgery in mild and moderate haemophilia A: a prospective national study in the Netherlands

Inhibitor development is currently the most severe complication in mild/moderate haemophilia A patients, causing increased bleeding tendency, hospitalization and mortality. It has been suggested that receiving high doses of factor VIII (FVIII) concentrates for surgical procedures is an important risk factor for inhibitor development in these patients. The current multicentre study aimed to determine prospectively the incidence of inhibitor development after intensive FVIII replacement therapy for surgical procedures in patients with mild/moderate haemophilia A. All consecutive patients with mild/moderate haemophilia A were included when they required at least 10 000 iu of FVIII concentrates (or 250 iu/kg) for 5 or more days for a surgical procedure. Potential clinical risk factors for inhibitor development and results of inhibitor tests were collected. Forty-six patients with a median age of 54 years (interquartile range, 40-59 years) were included in the study. F8 genotyping revealed 20 different missense mutations. Patients received either recombinant (65%) or plasma-derived FVIII concentrates (35%) by intermittent bolus injections (41%) or continuous infusion (57%). Two patients developed a low titre inhibitor post-operatively. The incidence of inhibitor development following intensive treatment for surgery in this unselected prospective cohort of mild/moderate haemophilia A patients was 4% (95% confidence interval, 0·5-14·8).

Pilot investigation of a novel testing strategy for bleeding in ventricular assist device recipients

A universal loss of von Willebrand factor (vWF) high-molecular-weight multimers (HMWM) has been demonstrated in continuous-flow left ventricular assist device (HeartMate II) recipients. However, no reliable clinical or laboratory predictors for an increased bleeding tendency in this patient population have been identified. This study evaluated the ability of a new automated latex particle-enhanced immunoturbidimetric vWF activity assay (ALPEIVA) to predict non-surgical bleeding risk in HeartMate II recipients. As part of a prospective multicenter trial, pre-surgical, 7-day, and 30-day post-implantation blood samples were collected from 24 patients. ALPEIVA-assessed vWF activities were compared among patients with and without non-surgical bleeding complications after HeartMate II implantation. Additional laboratory testing included factor VIII activity (FVIII:C), vWF antigen (vWFAg), vWF ristocetin cofactor activity (vWF:RCo), and vWF multimer analysis. All 24 patients had HMWM losses after HeartMate II implantation. Five patients (20%) developed non-surgical bleeding complications between 14 days and 6 months after HeartMate II implantation. Among various laboratory variables, only mean ALPEIVA/vWFAg ratios (referred to as the "bleeding ratio") were significantly lower in patients with clinically relevant bleeding (mean, 0.70 ± 0.06) compared with patients without bleeding (mean, 0.78 ± 0.09; p = 0.02) when measured at 30 days. The post-surgical bleeding ratio could potentially predict non-surgical bleeding risk and guide anti-platelet and anti-coagulation strategies in HeartMate II recipients.

Fibrinogen Šumperk II: Dysfibrinogenemia in an individual with two coding mutations

Fibrinogen—a 340-kDa glycoprotein—plays a crucial role in blood coagulation, platelet aggregation, wound healing, and other physiological processes. A mutation in fibrinogen may lead to congenital dysfibrinogenemia,a rare disease characterized by the functional deficiency of fibrinogen. About 580 cases of abnormal fibrinogens have been reported worldwide; thereof 335 cases in the fibrinogen Aa chain[1]. To our knowledge, only five cases of abnormal fibrinogens with two mutations [2–6] and one case of two different mutations in the same family [7] have been described earlier. A 52-year-old female was examined for bleeding. Routine hemostasis screening resulted in a diagnosis of dysfibrinogenemia. Functional testing revealed prolonged fibrin polymerization, prolonged lysis of the clot, abnormal fibrin morphology,and fibrinopeptides release. Genetic analysis showed two heterozygous nonsense mutations—previously described mutation AaGly13Glu and a novel mutation Aa Ser314Cys. The mutation Aa Gly13-Glu was found in her brother and niece, but there was no evidence in either of the mutation Aa Ser314Cys. While mutation Aa Gly13Glu is responsible for abnormal fibrinopeptide release and prolonged thrombin time, the novel mutation Aa Ser314Cys seems to affect fibrin morphology and fibrinolysis.

Protein Z Concentrations in Patients With Acute Leukemia

Protein Z (PZ) deficiency may induce bleeding as well as thrombosis. The aim of our study was to estimate the concentration of PZ in patients with acute leukemia. Plasma levels of PZ were determined in 76 patients with newly diagnosed acute leukemia ([AML], n = 50; acute lymphoblastic leukemia [ALL], n = 26) and 62 healthy participants. In the patients, mean plasma concentrations of PZ were statistically lower than in healthy individuals: AML (1.24 ± 0.11 μg/mL vs 1.58 ± 0.05 μg/mL P = .01) and ALL (1.19 ± 0.16 μg/mL vs 1.58 ± 0.05 μg/mL P = .01). Levels of PZ below the fifth percentile (0.873 μg/mL) of normal value distribution in control participants were found in 30% of patients with AML and ALL and in 3% of controls (P < .0001). In this AML subgroup, we found statistically significant correlation between episodes of bleeding and PZ level (P = .01). There was no such correlation in ALL group. The results suggest that PZ can be a cofactor associated with an increased bleeding tendency in patients with AML.

Defects in Glanzmann thrombasthenia and LAD-III (LAD-1/v) syndrome: the role of integrin β1 and β3 in platelet adhesion to collagen

AbstractPatients with Glanzmann thrombasthenia or Leukocyte Adhesion Deficiency-III syndrome (LAD-III or LAD-1/variant) present with increased bleeding tendency because of the lack or dysfunction of the fibrinogen receptor GPIIb/IIIa (integrin αIIbβ3), respectively. Although the bleeding disorder is more severe in LAD-III patients, classic aggregometry or perfusion of Glanzmann or LAD-III platelets over collagen-coated slides under physiologic shear rate does not discriminate between these 2 conditions. However, in a novel flow cytometry-based aggregation assay, Glanzmann platelets were still capable of forming small aggregates upon collagen stimulation, whereas LAD-III platelets were not. These aggregates required functional GPIa/IIa (integrin α2β1) instead of integrin αIIbβ3, thus explaining the clinically more severe bleeding manifestations in LAD-III patients, in which all platelet integrins are functionally defective. These findings provide genetic evidence for the differential requirements of platelet integrins in thrombus formation and demonstrate that correct integrin function assessment can be achieved with a combination of diagnostic methods.

Platelet counts and mean platelet volume in association with serum magnesium in maintenance hemodialysis patients.

Introduction: Platelet dysfunction is responsible for increased bleeding tendency of chronic renal failure patients. Magnesium (Mg) retention can occur in patients on maintenance hemodialysis.Objectives: Studies concerning the impact of magnesium disturbances on platelet counts and mean volume in hemodialysis are quiet scarce. Patients and Methods: A total of 36 (f= 15, m= 21), HD patients were included. The mean patients’ age was 46 (±16) years. The mean length of time patients had received hemodialysis was 32 (±36) (median: 19) months. Results: The mean platelet count was 165 (±70) (median: 163) [x103μ/L]. The mean platelet volume was 9 (±1) (median: 9.2) fl. The mean Mg was 9.2 ±1.4 (median: 2.4) mg/dl. In this study a significant inverse correlation of platelet count with mean platelet volume (r= -0.39, p= 0.017) was seen. A significant inverse correlation of serum Magnesium with mean platelet volume (r= -0.37, p= 0.025) was seen. There was not significant association of serum Mg and PLT count was found too (p> 0.05). Conclusion: A significant inverse correlation of serum Magnesium with mean platelet volume needs further investigations to clarify the clinical significance of this finding.

Orthognathic surgery in Melnick–Needles-Syndrome. Case report and review of the literature

Melnick–Needles Syndrome (MNS) is a congenital syndrome associated with severe architectural disorder of the skeletal system that can cause significant effects on the craniofacial skeleton including poor aesthetics, impaired speech and masticatory problems. The authors report a case of a female patient who experienced masticatory constraint, hindered speech and functional problems related to a severe bite dysfunction due to the mandibular hypoplasia and the underlying bony architectural disorder associated with MNS. The patient underwent bilateral sagittal split osteotomy (BSSO) to correct her skeletal malocclusion and improve the characteristic aesthetics and speech. The inherent bony abnormalities with their altered collagen structure presented unique challenges to orthognathic surgery in this patient with an unpredictable bone healing process. In MNS patients a fragile trabecular bone structure and an increased bleeding tendency is thought to delay or forestall wound healing. The patient was treated successfully with BSSO and monocortical plate fixation following a well-established algorithm from orthodontic preparation to surgical protocol. A very satisfying outcome has been achieved, concerning functional rehabilitation and aesthetic improvement. A review of the literature revealed that little is known about bone regeneration and fracture healing in cases with MNS.

Pharmacology and cellular/molecular mechanisms of action of aspirin and Non-aspirin NSAIDs in colorectal cancer

Colorectal cancer (CRC) and colorectal adenomas have in common a dysfunctional adenomatous polyposis coli suppressor gene (APC). This allows for activation of the oncogenic Wnt/β-catenin pathway, resulting in cytosolic accumulation of β-catenin, its translocation to the nucleus and action as a cofactor for stimulation of gene transcription. Pharmacological approaches of CRC-chemoprevention are focused to prevention of this β-catenin-mediated oncogenic signalling. Among upregulated genes in tumour tissue is COX-2 which synthesises large amounts of PGE(2). PGE(2) inhibits apoptosis, acts proinflammatory and immunosuppressive and stimulates tumour angiogenesis and proliferation. In addition, COX-2 causes oxidation (activation) of cocarcinogens. Aspirin and non-aspirin NSAIDs inhibit COX-2, subsequent PGE(2) formation and action by transcriptional and non-transcriptional mechanisms. These also include inhibition of generation of sphingosine-1-phosphate, an amplifier of these reactions and stimulation of NSAID-induced gene (NAG-1) which acts as an inhibitor. Aspirin additionally acetylates COX-2, resulting in generation of 'aspirin-triggered' lipoxins (ATL), a new class of anti-inflammatory/antitumour compounds. COX-1 inhibition might also contribute to antitumour effects of aspirin, for example at low-dose aspirin. Experimental evidence suggests additional COX independent actions of aspirin and non-aspirin NSAIDs on oncogenic signalling. This includes modifications of transcription factors (NFκB), induction of apoptosis and DNA stabilization. In comparison to non-aspirin NSAIDs (sulindac, indomethacin) and coxibs (celecoxib), aspirin has the advantage of concomitant antiplatelet effects while NSAIDs rather have a thrombogenic potential. Though these actions of aspirin have to be balanced against an increased bleeding tendency, aspirin is currently the most attractive candidate for clinical CRC chemoprevention. Open questions, such as dose, (minimum) duration of treatment and the individual risk/benefit ratio are subjects of prospective randomized trials which are underway.

Novel platelet activation receptor CLEC-2: from discovery to prospects.

C-type lectin-like receptor 2 (CLEC-2) has been identified as a receptor for the platelet activating snake venom rhodocytin. CLEC-2 elicits powerful platelet activation signals in conjunction with Src, Syk kinases, and phospholipase Cγ2, similar to the collagen receptor glycoprotein (GP) VI/FcRγ-chain complex. In contrast to GPVI/FcRγ, which initiates platelet activation through the tandem YxxL motif immunoreceptor tyrosine-based activation motif (ITAM), CLEC-2 signals via the single YxxL motif hemi-ITAM. The endogenous ligand of CLEC-2 has been identified as podoplanin, which is expressed on the surface of tumour cells and facilitates tumour metastasis by inducing platelet activation. Studies of CLEC-2-deficient mice have revealed several physiological roles of CLEC-2. Podoplanin is also expressed in lymphatic endothelial cells as well as several other cells, including type I alveolar cells and kidney podocytes, but is absent from vascular endothelial cells. In the developmental stages, when the primary lymph sac is derived from the cardinal vein, podoplanin activates platelets in lymphatic endothelial cells by binding to CLEC-2, which facilitates blood/lymphatic vessel separation. Moreover, CLEC-2 is involved in thrombus stabilisation under flow conditions in part through homophilic interactions. However, the absence of CLEC-2 does not significantly increase bleeding tendency. CLEC-2 may be a good target protein for novel anti-platelet drugs or anti-metastatic drugs having therapeutic and preventive effects on arterial thrombosis and cancer, the primary causes of mortality in developed countries. In this article, we review the mechanisms of signal transduction, structure, expression, and function of CLEC-2.

Essential in vivo roles of the platelet activation receptor CLEC-2 in tumour metastasis, lymphangiogenesis and thrombus formation

We have recently identified C-type lectin-like receptor 2 (CLEC-2) as a receptor for the platelet activating snake venom rhodocytin. CLEC-2 elicits powerful platelet activation signals in conjunction with single YxxL motif in its cytoplasmic tail, Src, Syk kinases, and phospholipase Cγ2. An endogenous ligand of CLEC-2 has been identified as podoplanin, which is a membrane protein of tumour cells and facilitates tumour metastasis by inducing platelet activation. Studies of CLEC-2-deficient mice have revealed several physiological roles of CLEC-2. Podoplanin is also expressed in lymphatic endothelial cells. In the developmental stages, when the primary lymph sac is derived from the cardinal vein, podoplanin activates platelets in lymphatic endothelial cells, which facilitates blood/lymphatic vessel separation. Moreover, CLEC-2 is involved in thrombus stabilization under flow conditions in part through homophilic interactions. The absence of CLEC-2 does not significantly increase bleeding tendency, implying that CLEC-2 may be a good target protein for anti-platelet drugs in addition to anti-metastatic drugs.

Multiple Electrode Whole Blood Aggregometry, PFA-100, and In Vivo Bleeding Time for the Point-of-Care Assessment of Aspirin-Induced Platelet Dysfunction in the Preoperative Setting

Acquired platelet dysfunction due to aspirin ingestion may increase bleeding tendency during surgery. Thus, we examined the diagnostic accuracy of in vivo bleeding time (BT) and 2 platelet function assays for the preoperative assessment of a residual antiplatelet effect in patients treated with aspirin. Consecutive patients scheduled for surgery were prospectively enrolled in this study. The patients' last aspirin ingestion had occurred within the previous 48 hours before blood sampling in the "full aspirin effect" group, between 48 and 96 hours before in the "variable aspirin effect" group, and >96 hours before in the "recovered aspirin effect" group. The control group had not taken any aspirin. Multiple electrode aggregometry, platelet function analyzer (PFA)-100, and in vivo BT were performed to assess the effects of aspirin. One-way analysis of variance on ranks with a post hoc multiple-comparison procedure (Dunn) was used to detect differences among the groups. Categorical data were compared using the z test. Receiver operating characteristic (ROC) curves were created to determine the diagnostic accuracy of the platelet function assays investigated. The area under the ROC curve (AUC), sensitivity, and specificity of the assays were calculated. The level of statistical significance was set at P < 0.05. Three hundred ninety-four patients were included in the analysis (133 control and 261 aspirin-treated patients). All 3 methods were able to detect the antiplatelet effect of aspirin in the full aspirin effect group. Furthermore, no difference in the measurement values between the recovered aspirin effect and control group was found, irrespective of the assay performed. Measurement values in the variable aspirin effect group were different from those of the control group in the ASPItest using multiple electrode aggregometry and COL-EPI using PFA-100 but not in BT. ROC analysis showed the highest diagnostic accuracy in excluding the residual aspirin effect in the ASPItest (AUC 0.81, P < 0.001), followed by COL-EPI (AUC 0.78, P < 0.001) and BT (AUC 0.56, P = 0.05). The cutoff value of 53 U in the ASPItest excluded the effect of aspirin with a sensitivity of 88% and specificity of 71%. The full therapeutic antiplatelet effects of aspirin can be expected within 48 hours of the patient's last aspirin ingestion. Platelet function recovered in our study if aspirin cessation occurred >96 hours (4 days) before; thus, in these patients, preoperative platelet function testing is not useful. To quantify any residual aspirin effect in patients who ceased their intake of aspirin between 48 and 96 hours before surgery, the ASPItest might have the highest diagnostic accuracy.

Escitalopram and Ischemic Stroke: Causal or Chance Association?

Escitalopram and Ischemic Stroke: Causal or Chance Association?

Effects of aspirin and desmopressin on platelet reactivity in patients undergoing cardiac surgery with extracorporeal circulation

The effect of desmopressin on platelet function in patients with continued antiplatelet therapy undergoing cardiac surgery is discussed controversially. We assessed platelet reactivity in 86 patients undergoing elective coronary artery bypass grafting (CABG) under extracorporeal circulation. Twenty-nine of these patients were without preoperative antiplatelet therapy (group A), while 57 were treated with acetylsalicylic acid (ASA) 100 mg qd up to the day of surgery. Out of this cohort, 24 patients received no desmopressin perioperatively (group B), whereas 33 patients were treated with desmopressin 0.4 microg/kg after administration of protamine due to increased bleeding tendency (group C). Multiple electrode platelet aggregometry with arachidonic acid as agonist showed a marked decrease of platelet reactivity in patients without antiplatelet therapy immediately after extracorporeal circulation compared to preoperative control (375 ± 227 vs. 749 ± 330 AU*min, p<0.001). Platelet reactivity recovered to preoperative controls in group A at 24 hours after protamine administration (662 ± 295 AU*min). Platelet reactivity in patients on ASA was not decreased further after extracorporeal circulation (group B: 197 ± 126 vs. 251 ± 203 AU*min, p=0.14; group C: 212 ± 100 vs. 245 ± 248 AU*min, p=0.43) and improved significantly within 24 hours. A statistically significant effect of desmopressin, however, could not be determined (group B: 392 ± 223 AU*min; group C: 439 ± 324 AU*min at 24 hours after protamine, p=0.63 for between-subjects contrast). Our data suggest that desmopressin does not affect platelet reactivity in patients on ASA undergoing CABG and is, therefore, not useful in this clinical setting.

Association between phenotype and genotype in carriers of haemophilia A

Female carriers of haemophilia might suffer from increased bleeding tendency therefore the assessment of the bleeding risk is very important for improving care. This single-centre study documents the occurrence of bleedings in 46 carriers of haemophilia A including bleeding after tooth extraction (77%), easy bruising (67%), postsurgical bleeding (61%), menorrhagia (50%) or prolonged postpartum bleeding (43%). The F8 gene mutation of all 46 carriers (median age: 36.5 years, 15-80 years; mean FVIII:C activity: 59 ± 24.45%; normal range: 64-167%) was determined, and family history of haemophilia was recorded. For analysis, the bleeding tendency of the carriers was differentiated by severity into three groups. There was no statistically significant difference of FVIII:C between these groups. However, a correlation was found between the severity of bleeding tendency and the type of F8 gene mutation (P < 0.05) as well as the severity of haemophilia in affected male relatives (P < 0.0005). Results show that even carriers with a FVIII:C activity as high as 50-60% are at increased risk of bleeding. Incidence and intensity of bleeding symptoms of haemophilia A carriers are high and correlated with the phenotype of the male haemophilic relative and the underlying F8 gene mutation.

Thromboelastometry (ROTEM®) in children: age-related reference ranges and correlations with standard coagulation tests

BackgroundThe small sample volume needed and the prompt availability of results make viscoelastic methods like rotational thromboelastometry (ROTEM®) attractive for monitoring coagulation in small children. However, data on reference ranges for ROTEM® parameters in children are scarce. MethodsFour hundred and seven children (ASA I and II) undergoing elective surgery were recruited for this prospective, two-centre, observational study. Subjects were grouped as follows: 0–3, 4–12, 13–24 months, 2–5, 6–10, and 11–16 yr. Study objectives were to establish age-dependent reference ranges for ROTEM® assays, analyse age dependence of parameters, and compare ROTEM® data with standard coagulation tests. ResultsData from 359 subjects remained for final analysis. Except for extrinsically activated clot strength and lysis, parameters for ROTEM® assays were significantly different among all age groups. The most striking finding was that subjects aged 0–3 months exhibited accelerated initiation (ExTEM coagulation time: median 48 s, Q1–Q3 38–65 s; P=0.001) and propagation of coagulation (α angle: median 78o, Q1–Q3 69–84o; P<0.001) and maximum clot firmness (median 62 mm, Q1–Q3 54–74 mm), although standard plasma coagulation test results were prolonged (prothrombin time: median 13.2 s, Q1–Q3 12.6–13.6 s; activated partial thromboplastin time: median 42 s, Q1–Q3 40–46 s). Lysis indices of <85% were observed in nearly one-third of all children without increased bleeding tendency. Platelet count and fibrinogen levels correlated significantly with clot strength, and fibrinogen levels correlated with fibrin polymerization. ConclusionsReference ranges for ROTEM® assays were determined for all paediatric age groups. These values will be helpful when monitoring paediatric patients and in studies of perioperative coagulation in children.

Mutation and ADAMTS13-dependent modulation of disease severity in a mouse model for von Willebrand disease type 2B

AbstractVon Willebrand disease (VWD)–type 2B originates from a gain-of-function mutation in von Willebrand factor (VWF), resulting in enhanced platelet binding. Clinical manifestations include increased bleeding tendency, loss of large multimers, thrombocytopenia, and circulating platelet aggregates. We developed a mouse model to study phenotypic consequences of VWD-type 2B mutations in murine VWF: mVWF/R1306Q and mVWF/V1316M. Both mutations allow normal multimerization but are associated with enhanced ristocetin-induced platelet aggregation, typical for VWD-type 2B. In vivo expression resulted in thrombocytopenia and circulating aggregates, both of which were more pronounced for mVWF/V1316M. Furthermore, both mutants did not support correction of bleeding time or arterial vessel occlusion in a thrombosis model. They further displayed a 2- to 3-fold reduced half-life and induced a 3- to 6-fold increase in number of giant platelets compared with wild-type VWF. Loss of large multimers was observed in 50% of the mice. The role of ADAMTS13 was investigated by expressing both mutants in VWF/ADAMTS13 double-deficient mice. ADAMTS13 deficiency resulted in more and larger circulating platelet aggregates for both mutants, whereas the full multimer range remained present in all mice. Thus, we established a mouse model for VWD-type 2B and found that phenotype depends on mutation and ADAMTS13.

Use of Serotonergic Antidepressants and Bleeding Risk in Orthopedic Patients

Selective serotonin reuptake inhibitors have been associated with an increased bleeding tendency. Information on the impact of a possible impaired hemostasis associated with the perioperative use of selective serotonin reuptake inhibitors is limited. This study aimed to determine the association between the perioperative use of selective serotonin reuptake inhibitors and the amount of blood loss during surgery and perioperative transfusion requirements. The authors conducted a retrospective cohort study among patients who underwent elective primary total hip arthroplasty in two hospitals from the period of July 1, 2004 until July 1, 2008. The index group included all users of both serotonergic and nonserotonergic antidepressants. The reference group included a random sample (ratio 1:3) of nonusers. The primary outcome was the amount of intraoperative blood loss. The requirement for blood transfusion was a secondary outcome. The outcomes were adjusted for confounding factors (comorbidity and comedication) using regression techniques. The index group included 66 users of serotonergic and 29 users of nonserotonergic antidepressants, and the reference group included 285 patients. After adjustment for confounding factors, mean blood loss during surgery was significantly higher in the users of selective serotonin reuptake inhibitors when compared with the reference group: 95 ml (95% CI 9-181). Mean blood loss in the users of nonserotonergic antidepressants did not differ from the reference group. Users of antidepressants did not have a higher risk for transfusion. Patients undergoing total hip arthroplasty who continue the use of serotonergic antidepressants show a significantly higher, but clinically unimportant, intraoperative blood loss, without an increase in perioperative transfusion requirements.

COU254, a specific 3-carboxamide-coumarin inhibitor of coagulation factor XII, does not protect mice from acute ischemic stroke

BackgroundAnticoagulation is an important means to prevent from acute ischemic stroke but is associated with a significant risk of severe hemorrhages. Previous studies have shown that blood coagulation factor XII (FXII)-deficient mice are protected from pathological thrombus formation during cerebral ischemia without bearing an increased bleeding tendency. Hence, pharmacological blockade of FXII might be a promising and safe approach to prevent acute ischemic stroke and possibly other thromboembolic disorders but pharmacological inhibitors selective over FXII are still lacking. In the present study we investigated the efficacy of COU254, a novel nonpeptidic 3-carboxamide-coumarin that selectively blocks FXII activity, on stroke development and post stroke functional outcome in mice.MethodsC57Bl/6 mice were treated with COU254 (40 mg/kg i.p.) or vehicle and subjected to 60 min transient middle cerebral artery occlusion (tMCAO) using the intraluminal filament method. After 24 h infarct volumes were determined from 2,3,5-Triphenyltetrazoliumchloride(TTC)-stained brain sections and functional scores were assessed. Hematoxylin and eosin (H&E) staining was used to estimate the extent of neuronal cell damage. Thrombus formation within the infarcted brain areas was analyzed by immunoblot.ResultsInfarct volumes and functional outcomes on day 1 after tMCAO did not significantly differ between COU254 pre-treated mice or untreated controls (p > 0.05). Histology revealed extensive ischemic neuronal damage regularly including the cortex and the basal ganglia in both groups. COU254 treatment did not prevent intracerebral fibrin(ogen) formation.ConclusionsCOU254 at the given concentration of 40 mg/kg failed to demonstrate efficacy in acute ischemic stroke in this preliminary study. Further preclinical evaluation of 3-carboxamide-coumarins is needed before the antithrombotic potential of this novel class of FXII inhibitors can be finally judged.

A Large Pedigree with Von Willebrand Disease (VWD) Caused by a Novel Unifying Mutation (M1304R) Exhibits Significant Laboratory and Clinical Phenotypic Heterogeneity.

Abstract 542 We have characterized a pedigree with VWD that exhibits significant phenotypic variability. The pedigree is composed of 24 Caucasian members and includes 12 individuals diagnosed with VWD, based on abnormal VWF levels and the presence of mucocutaneous bleeding. The pattern of transmission is autosomal dominant. Mutational analysis of exon 28 of the VWF locus was performed in all individuals. A novel single base T&gt;G substitution at position 3911 that predicts a methionine to arginine (M1304R) change in the A1 domain of the mature VWF molecule was detected in all affected individuals, and segregates with the bleeding phenotype. Sequence of the entire coding region of VWF in 1 affected and 1 non-affected member did not show any additional mutations. Von Willebrand factor antigen levels (VWF:Ag) and von Willebrand factor ristocetin cofactor activity (VWF:RCo) in the affected individuals are variable with a mean of 21.9 IU/dL (range 11 to 30) and 11.18 IU/dL (range 3 to 17) respectively. Platelet counts in all individuals of the pedigree are within the normal range. While bleeding scores in non-affected members are below 2 (no bleeding phenotype), they range from 3 to 7 among mutation positive individuals (indicating a wide variation in bleeding symptoms). Multimer analyses also show significant variability, with some individuals having a full range of multimer sizes and others only having low and medium size molecular weight multimers. This loss of high molecular weight multimers does not appear to correlate with increased bleeding tendency. We also studied, by ELISA, plasma from affected and non-affected individuals of the pedigree using a recently developed llama antibody (nanobody AU/VWFa-11) which recognizes 2B and/or 2M mutations of the A1 domain. A ratio (“activation factor”) that represents the relative amount of mutant VWF in patient plasma was generated between patients nanobody-bound VWF and that derived from normal pooled plasma. Nanobody binding in the patients was greater than in the unaffected family members, but varied considerably among affected members, with “activation factors” that range from 1.1 to 4.9. Since all individuals exhibit the same mutation this variation is not likely due to differences in binding affinity of patient VWF to the nanobody nor is it due to the difference in VWF concentration because the ratios are independent of VWF concentration. The variation in “activation factor” suggests that plasma VWF in the affected individuals contains different amount of mutant protein, and this may account for the wide range of bleeding tendencies among these patients. Expression of the M1304R VWF mutant in HEK293 cells show decreased secretion. It also fails to form storage granules, and the VWF secreted lacks high molecular weight multimers. When co expressed with the wild-type allele, total VWF secretion is decreased, multimerization is nearly normalized but still no storage granules are formed in HEK293 cells, suggesting that this particular mutation affects multimerization and trafficking. In summary, we have carefully characterized a family with VWD caused by a unifying mutation that exhibits significant phenotypic variability, which cannot be predicted by current availabel techniques. In order to further understand the clinical and laboratory variability of VWD, it is important to develop new technologies to quantify amounts of mutant VWF in plasma from VWD patients. Disclosures: No relevant conflicts of interest to declare.