The Jamaican Haemophilia Registry: Describing the burden of disease.

INTRODUCTION: Jamaica has an estimated 200 persons with haemophilia (PWH), who face significant constraints in access to specialized haemophilia care, including access to clotting factor concentrates. AIM: The aim of this paper is to establish the current burden of disease in PWH in Jamaica. METHODS: PWH were enrolled through the University Hospital of the West Indies, Jamaica. The impact of haemophilia was assessed using a comprehensive battery of heath outcome measures that included the following: laboratory, clinical information and validated outcome measures of joint structure and function, activity, and health-related quality of life (HRQoL) to provide a health profile of the Jamaican haemophilia population. RESULTS: In all, 45 PWH were registered (mean age: 29, range: 0.17-69 years), including 13 children (<18 years of age) and 32 adults. In this sample, 41 had haemophilia A (30 severe) and 4 had haemophilia B (3 severe); 10 patients with haemophilia A were inhibitor positive. The results indicate that adults with haemophilia in Jamaica have significant joint damage: mean Haemophilia Joint Health Score (HJHS) = 42.1 (SD = 17.3); moderate activity levels - mean Haemophilia Activities List (HAL) score = 64.8 (SD = 17.8); and low HRQoL scores - mean Haemo-QoL-A score = 62.3 (SD = 19.4). Results for children are also reported but should be interpreted with caution due to the small sample size. CONCLUSIONS: There is a very high burden of disease in PWH in Jamaica. The health profiles reported in this paper are an essential first step in advocating for a multidisciplinary Comprehensive Care Program for assessment and care of PWH in Jamaica.

Normal range of bleeding scores for the ISTH-BAT: adult and pediatric data from the merging project.

Bleeding Assessment Tools (BATs) have been developed to aid in the standardized evaluation of bleeding symptoms. The Vicenza Bleeding Questionnaire (BQ), published in 2005, established a common framework and scoring key that has undergone subsequent modification over the years, culminating in the publication of the ISTH-BAT in 2010. Understanding the normal range of bleeding scores is critical when assessing the utility of a BAT. Within the context of The Merging Project, a bioinformatics system was created to facilitate the merging of legacy data derived from four different (but all Vicenza-based) BATs; the MCMDM1-VWD BQ, the Condensed MCMDM-1VWD BQ, the Pediatric Bleeding Questionnaire and the ISTH-BAT. Data from 1040 normal adults and 328 children were included in the final analysis, which showed that the normal range is 0-3 for adult males, 0-5 for adult females and 0-2 in children for both males and females. Therefore, the cut-off for a positive or abnormal BS is ≥4 in adult males, ≥6 in adult females and ≥3 in children. This information can now be used to objectively assess bleeding symptoms as normal or abnormal in future studies.

Diannexin, an annexin A5 homodimer, binds phosphatidylserine with high affinity and is a potent inhibitor of platelet-mediated events during thrombus formation.

BACKGROUND: Shielding of procoagulant phosphatidylserine (PS) with annexin A5 attenuates thrombosis, but annexin A5 (35.7 kDa) is rapidly cleared from the circulation. In contrast, Diannexin, a 73.1 kDa homodimer of annexin A5, has an extended half-life. OBJECTIVES: To quantify the affinity of Diannexin for PS, examine its interaction with activated platelets and determine its effects on platelet-mediated events during thrombus formation. METHODS: The affinities of Diannexin and annexin A5 for PS-containing lipid bilayers were compared using surface plasmon resonance, and binding to activated platelets was assessed by flow cytometry. Calibrated automated thrombography and thromboelastography were employed to study the effects of Diannexin on thrombin generation and platelet-fibrin clot formation, respectively, whereas intravital videomicroscopy was used to examine its effect on platelet accumulation and activation after laser-induced injury to murine cremaster arterioles, and a tail tip bleeding model was used to explore its effects on hemostasis. RESULTS: Diannexin and annexin A5 bind PS with K(D) values of 0.6 and 5 nm, respectively, and both bind to the same subpopulation of PS-exposing platelets. Diannexin inhibited thrombin generation and platelet-fibrin clot formation in vitro at 10 nm (P<0.05-0.001 compared with control), and reduced platelet accumulation at 1 µg g(-1) (P<0.05) and activation at 0.25 µg g(-1) (P<0.001) in experimentally induced arterial thrombi in mice while increasing blood loss at 1 µg g(-1) (P<0.01). CONCLUSIONS: Diannexin binds to PS with high affinity and is a potent inhibitor of platelet-mediated events during thrombus formation.

Expanded phenotype-genotype correlations in a pediatric population with type 1 von Willebrand disease.

BACKGROUND: Recent phenotype-genotype studies have provided valuable insights into the pathophysiology of type 1 von Willebrand disease (VWD); however, no study has examined an exclusively pediatric cohort. OBJECTIVES: To describe phenotype-genotype correlations in a selected pediatric cohort with a historical diagnosis of type 1 VWD, using first-degree family members as controls. METHODS: Comprehensive phenotypic assessment included standard assays of von Willebrand factor (VWF) level and function, bleeding score, desmopressin response, VWF propeptide (VWFpp) level, and platelet-derived VWF mRNA level. RESULTS: Fourteen VWF mutations were identified in 17 of 23 index cases (ICs) (aged 5-17 years), including four that were previously unreported (L60P, nt1658 insT, Q1388X, and C2237F). VWFpp levels were lower in ICs than in unaffected controls (median 49 vs. 86 U dL(-1) , P < 0.0001). A VWFpp/VWF antigen ratio of > 1.6 was observed in eight of nine ICs with a suboptimal response to desmopressin, including four of four with the R1205H (Vicenza) mutation (median 7.9), and three of four IC with the R1315C mutation (median 1.9). The R1315C mutation was also associated with a reduced absolute VWFpp level (median 32 U dL(-1) ), a previously unreported finding. The amount of platelet-derived VWF mRNA was significantly reduced in individuals with nonsense mutations. CONCLUSIONS: Increased VWF clearance and intracellular retention are important mechanisms underlying type 1 VWD in pediatric patients, concordant with the observations of larger, predominantly adult, cohort studies. Additionally, in some patients, nonsense-mediated decay of mutant mRNA transcripts may be contributory. Several mechanisms underlie the variable phenotype associated with the R1315C mutation. The potential utility of VWFpp as an independent marker of VWF biosynthesis and release warrants further research.

Quantitation of bleeding symptoms in children with von Willebrand disease: use of a standardized pediatric bleeding questionnaire.

SUMMARY BACKGROUND: Excessive bruising and mucocutaneous bleeding are frequent presenting symptoms in childhood. A detailed bleeding history can distinguish children who may have an inherited bleeding disorder from those who are normal. There is a lack of standardization of such history taking in pediatric practise. OBJECTIVES: To assess the performance of a Pediatric Bleeding Questionnaire (PBQ), an adaptation of a standardized adult bleeding questionnaire and score that includes pediatric-specific bleeding symptoms, in a cohort of children with von Willebrand disease (VWD). PATIENTS/METHODS: Bleeding scores were determined by interview, for children with a previous diagnosis of VWD and a control group of unaffected siblings. RESULTS: Bleeding scores were obtained for 100 children with VWD, median age 10.9 years (range, 0.8-17.8 years), and 21 unaffected siblings. Median bleeding score in children with VWD was 7.0 (range, 0-29) and in the control group was 0 (range, -1-2). Bleeding score varied within and between each VWD type: definite type 1, n = 40, median, 9.0 (range, 2-18); possible type 1, n = 38, median, 2.0 (0-15); type 2, n = 6, median, 14.0 (3-17); and type 3, n = 16, median, 12.0 (4-29). Bleeding scores in affected children correlated with age (Spearman's correlation coefficient, 0.35; P = 0.0004). The most frequent clinically significant bleeding symptoms were surgical bleeding, bleeding after tooth extraction and menorrhagia. Post-circumcision bleeding, cephalohematoma, macroscopic hematuria and umbilical stump bleeding were clinically significant in 32% (of circumcised males), 4%, 4% and 3% of children, respectively. CONCLUSIONS: The PBQ provides a standardized quantitation of bleeding severity in children with VWD.

In vitro and in vivo stability of diluted recombinant factor VIII for continuous infusion use in haemophilia A.

Factor VIII (FVIII) replacement by continuous infusion (CI) is used postoperatively or after significant bleeding. For young paediatric patients, CI may require FVIII dilution. Variable stabilities of diluted full-length recombinant FVIII Kogenate FS (KG-FS) have been reported under different storage conditions. We investigated the recovery and stability of diluted KG-FS in vitro and in vivo. Kogenate FS was diluted to 50-120 U mL(-1) and its recovery and stability in glass vials or polypropylene syringes was determined. Furthermore, stability of KG-FS diluted to 80 U mL(-1)'administered' via single- and double-pump mock CI systems was tested. Finally, the in vivo stability of KG-FS diluted to approximately 60 U mL(-1) and administered postsurgically by CI with the double-pump to a paediatric patient with severe haemophilia A undergoing implantable venous access device placement was investigated. Initial KG-FS dilution resulted in a 10-20% FVIII loss; a further 25-30% loss occurred over 72 h in vials or syringes. With the double-pump, 1 h recovery was 35%, increasing to 80% by 24 h; the initial losses were because of the Y-infusion of a 10-fold larger volume of saline concomitantly with the FVIII. In vivo, CI resulted in stable FVIII activity levels within the target range. These in vitro results are important for the generation of CI guidelines for diluted KG-FS in the paediatric haemophilic population. That FVIII losses occur upon dilution and with the double-pump does not preclude use of diluted KG-FS. Indeed, stable FVIII levels were maintained when diluted KG-FS was administered by CI with the double-pump to a paediatric patient postsurgically.

Persistence of procoagulant surface expression on activated human platelets: involvement of apoptosis and aminophospholipid translocase activity.

BACKGROUND: Activated platelets express a procoagulant surface when the asymmetric distribution of membrane phospholipids is scrambled, leading to phosphatidylserine (PS) exposure. PS expression, associated with apoptosis in nucleated cells, would be expected to be reversed by aminophospholipid translocase (APLT) activity. OBJECTIVE: To determine whether the procoagulant surface of activated platelets persists after it forms; to examine whether PS expression on platelets is associated with loss of mitochondrial inner membrane potential (DeltaPsi(m)), a hallmark of apoptosis; and to investigate the role of APLT in persistence of PS expression. METHODS: Platelets were stimulated with thrombin, collagen, a combination of both, or the Ca(2+)-ionophore A23187. Up to 4 h after activation, procoagulant surface expression was measured by annexin A5 binding by flow cytometry and by a prothrombinase assay. Flow cytometry was also used to measure PS expression concurrently with DeltaPsi(m) collapse, using CMXRos. APLT activity in annexin A5-negative and -positive platelets was measured flow cytometrically as the percent of 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl]-sn-glycero-3-phosphatidylserine (NBD-PS) translocated from the outer to the inner membrane leaflet. RESULTS AND CONCLUSIONS: Procoagulant surface expression on activated platelets persisted in vitro for at least 4 h; if such persistence occurs in vivo, there are important implications for the propagation of thrombosis. With the physiological stimuli, only 10-20% of the activated platelets expressed PS on their surface, and of these, only a portion exhibited DeltaPsi(m) collapse, indicating that PS expression can be associated with platelet apoptosis, but can also occur independently. APLT activity was very low in the PS-expressing platelet subpopulation for up to 4 h after activation, indicating that the persistence of a procoagulant surface may be attributed, at least in part, to this reduced APLT activity.

Triple heterozygosity in the integrin alphaIIb subunit in a patient with Glanzmann's thrombasthenia.

We report triple heterozygosity in the integrin alpha(IIb) subunit in a 5-year-old Canadian girl with Glanzmann's thrombasthenia. The patient has a severe bleeding history possibly aggravated by low VWF suggestive of associated type 1 von Willebrand's disease. Platelet aggregation was absent or severely reduced for all physiologic agonists. Flow cytometry showed an approximately 4% residual surface expression of alpha(IIb)beta(3). Western blotting confirmed a low platelet expression of both subunits. PCR-SSCP and direct sequencing showed no abnormalities in the beta(3) gene, but revealed a G-->A transition at a splice site [IVS 19 (+1)] of exon 19 in the alpha(IIb) gene. Of maternal inheritance, the splice site mutation was associated with intermediate levels of alpha(IIb)beta(3) in carriers. Unexpectedly, two G-->A transitions were detected in exon 29 of the alpha(IIb) gene and led to V(951)-->M and A(958)-->T amino acid substitutions. Family studies using restriction enzymes showed that both exon 29 mutations were paternal in origin and cosegregated across three generations. Transient expression in which mutated alpha(IIb) was cotransfected with wild-type beta(3) in COS-7 cells showed that V(951)-->M gave a much reduced surface expression of alpha(IIb)beta(3) and a block in the maturation of pro-alpha(IIb). In contrast, the A(958) substitution appeared to be a novel polymorphism. Our studies highlight an unusual mixture of defects giving rise to severe bleeding in a child and describe the first pathological missense mutation affecting a C-terminal residue of the calf-2 domain of alpha(IIb).

Procoagulant surface exposure and apoptosis in rabbit platelets: association with shortened survival and steady-state senescence.

BACKGROUND: The signal(s) for removal of senescent platelets from the circulation are not fully understood; phosphatidylserine (PS) expression on platelets and another marker of apoptosis, loss of mitochondrial inner membrane potential (DeltaPsim), have been implicated in platelet clearance. OBJECTIVE: To investigate whether shortened platelet survival and steady-state platelet senescence are associated with increased surface exposure of PS and DeltaPsim collapse. METHODS: Survival of in-vitro biotinylated rabbit platelets treated with thrombin or Ca(2+)-ionophore A23187 was tracked by flow cytometry after injection. Steady-state platelet senescence was investigated by infusing biotin to label a platelet cohort. PS expression and DeltaPsim of in-vitro biotinylated platelets and of the aging platelet cohort biotinylated in-vivo were measured by flow cytometry using annexin V-FLUOS and the DeltaPsim-sensitive dye CMXRos, respectively. RESULTS: Although PS expression, DeltaPsim and survival of thrombin-degranulated platelets were similar to those of control platelets, increasing concentrations of A23187 caused increased surface exposure of PS and progressive shortening of platelet survival; only one-sixth of PS-expressing platelets also exhibited DeltaPsim loss. The cohort of senescent, biotinylated platelets remaining in the circulation at 96 h had increased exposure of PS and collapsed DeltaPsim; of the 17% of PS-expressing platelets, one-third did not exhibit DeltaPsim loss. There was also an increase in platelets with collapsed DeltaPsim but not expressing PS. CONCLUSIONS: Platelets with shortened survival and senescent platelets have increased surface exposure of PS, that may be involved in their clearance. PS expression can occur independently of DeltaPsim collapse and conversely, in aged platelets, DeltaPsim loss can occur independently of PS expression.

The activity of the von Willebrand factor cleaving protease ADAMTS-13 in newborn infants.

BACKGROUND: Unusually large von Willebrand factor (VWF) multimers have been observed in patients with thrombotic microangiopathies (TMA), and absence of the VWF cleaving protease ADAMTS-13 activity is considered to be involved in the etiology of TMA. Increased amounts of large multimers of VWF have also been identified in neonates. OBJECTIVE: We assessed ADAMTS-13 activity in healthy neonates, children and adults to establish baseline levels. PATIENTS AND METHODS: Cord blood was collected from 38 full-term newborns; venous samples were taken from 15 neonates on day 2-3 of life. Seventeen children, 24 healthy adults and seven patients with TMA were studied as well. ADAMTS-13 activity was quantified by the binding of the subjects' plasma VWF to collagen before and after enzyme activation. The multimer distribution of VWF was also determined. RESULTS: Neonates and children had percentage ADAMTS-13 activity similar to adults. However, two groups were apparent in the cord blood samples: while 28/38 newborns had percentage activity within the normal range of healthy adults (102 +/- 3.0%), 10 had significantly lower percentage activity (53 +/- 1.1%; P < 0.0001) that normalized by day 2-3. The VWF multimer distribution was the same in all cord blood samples and was not different compared with children and adults. High-molecular-weight VWF multimers were significantly increased in the 2-3-day-old neonates and in TMA patients. CONCLUSIONS: Although ADAMTS-13 activity was similar in neonates compared with adults, 26% of neonates had mildly reduced activity. Further studies are needed to investigate the complex interaction of VWF production and secretion with its size control by ADAMTS-13 in different age groups.

Concurrent measurement of the survival of two populations of rabbit platelets labeled with either two PKH lipophilic dyes or two concentrations of biotin.

BACKGROUND: To avoid radioisotopic labeling and permit comparison of the survival of two platelet populations concurrently in one animal, we compared simultaneous recoveries and survival times of homologous rabbit platelets labeled in vitro with the lipophilic dyes PKH26 (red fluorescing) and PKH67 (green fluorescing) and with two levels of biotin (low, 1 microg/ml; high, 10 microg/ml). METHODS: Blood samples were drawn up to 96 h postinfusion and analyzed by flow cytometry. Biotin-labeled samples were incubated with phycoerythrin-streptavidin before analysis. RESULTS: Recovery of PKH26-labeled platelets at 1 h was lower (37.5%) than that of PKH67-labeled platelets (47.3%; P < 0.001). Platelet survival times were 62.4 and 61.9 h. Recoveries at 1 h of platelets labeled with two levels of biotin were similar (86.6% and 84.6%) and greater than those of PKH-labeled platelets (P < 0.001). Survival of platelets labeled with biotin did not differ (low, 83.3 h; high, 85.2 h) and was longer than for PKH-labeled platelets (P < 0.01). Labeling methods did not activate platelets (measured by P-selectin expression), nor did they affect platelet responses to adenosine diphosphate (ADP), collagen, or thrombin. CONCLUSIONS: Labeling with two levels of biotin is superior to labeling with PKH dyes, and is useful for measuring concurrently the survival of two differing platelet populations.

Blood platelet adhesion to protein studied by on-line acoustic wave sensor.

The attachment of blood platelets to the surface of bare and protein-coated thickness-shear mode acoustic wave devices operating in a flow-through configuration has been studied. Platelets in washed from bind to the gold electrodes of such sensors, but the resulting frequency shifts are far less than predicted by the conventional mass-based model of device operation. Adherence to albumin and various types of collagen can be produced by on-line introduction of protein or by a pre-coating strategy. Differences in attachment of platelets to collagen types I and IV and the Horm variety can be detected. Platelets attached to collagen yield an interesting delayed, but reversible signal on exposure to a flowing medium of low pH. Scanning electron microscopy of sensor surfaces at various time points in this experiment reveals that originally intact platelets are eventually destroyed by the high acidity of the medium. The reversible frequency is attributed to the presence of removable platelet granular components at the sensor-liquid interface.

von Willebrand disease in a pediatric-based population--comparison of type 1 diagnostic criteria and use of the PFA-100 and a von Willebrand factor/collagen-binding assay.

Definitive diagnosis of type 1 von Willebrand Disease (VWD) remains a problem. Provisional consensus guidelines for the diagnosis of definite and possible type 1 VWD were prepared by the Scientific Subcommittee on von Willebrand factor (VWF) of the Scientific and Standardization Committee (SSC) of the International Society on Thrombosis and Haemostasis (ISTH) during the 1996 annual meeting for the specific purpose of further evaluation in retrospective and prospective studies by a Working Party on Diagnostic Criteria (1996 Annual Report of the SSC/ISTH Subcommittee on VWF). In the first phase of this study, we compared 2 definitions of type 1 VWD. each with 3 criteria: significant bleeding history, laboratory investigations, and family history. Using the ISTH consensus guidelines for type 1 VWD definition, significantly fewer patients were diagnosed with definite type 1 disease as compared to our "in house" Hospital for Sick Children (HSC) criteria (4 vs. 31). While we recognize that the provisional ISTH consensus guidelines were not intended for clinical use, we believe that the results of our studies are of interest and will assist in any future refinements to the ISTH guidelines. In the second phase of this study, we investigated the utility of 2 new tests, a laboratory screening test and a functional test, for VWD in our well characterized, pediatric-based population. The Platelet Function Analyzer (PFA-100) provides an in vitro measure of primary hemostasis under conditions of high shear, using disposable cartridges containing collagen and either epinephrine or ADP. All tested subjects with types 2 or 3 VWD had prolonged PFA-100 closure times (CTs) with both cartridge types (n = 17) and prolonged bleeding times (n = 14). In subjects with definite type 1 VWD, 20/24 (83%) had prolonged CTs with the collagen/ADP cartridge (19/24 (79%) with collagen/epinephrine), compared with 7/26 (27%) with prolonged bleeding times. In subjects with definite types 1, 2, or 3 VWD, collagen/ADP CTs were abnormal in 37/41 subjects, giving an overall sensitivity of 90%. With this high sensitivity, the PFA-100 is a better screening test for VWD than the bleeding time. We also tested a VWF collagen-binding assay (VWF:CBA) as a functional test for VWF, in comparison with the more routinely-used ristocetin cofactor assay (VWF:RC0). The VWF:CBA is based on an ELISA technique, which has the potential to be more reproducible than the VWF:RC0. We found that the VWF:CBA detected 43/49 (88%) subjects with definite types 1, 2, or 3 VWD, performing as well as the VWF:RC0, that detected 42/48 (88%). We also showed that, used in conjunction with VWF antigen levels, the VWF:CBA may be useful in classification of VWD subtypes.