Women and inherited bleeding disorders - A review with a focus on key challenges for 2019.

The area of women and inherited bleeding disorders has undergone quick expansion in recent years. More patients are being identified and expertise to diagnose and manage these patients is now essential for practising physicians. Programs to help educate and empower patients and caregivers are now in place. Common inherited bleeding disorders affecting women include von Willebrand disease (VWD), inherited platelet disorders, and rare inherited bleeding disorders such as factor VII (FVII) deficiency and factor XI (FXI) deficiency. Specific clinical tools have been developed to help clinicians and patients screen for the presence of these bleeding disorders in both adult and pediatric populations. Affected women can experience heavy menstrual bleeding and resulting iron deficiency anemia, postpartum hemorrhage, and hemorrhagic ovarian cysts which need to be properly managed. Excessive bleeding can adversely affect quality of life in these women. Front line therapy for bleeding in mild cases focuses on the use of non-specific hemostatic agents such as DDAVP ®, tranexamic acid and hormonal agents but specific factor replacement and/or blood products may be required in more severe cases, in severe bleeding or as second line treatment when bleeding is not responsive to first line agents. Iron status should be optimised in these women especially in pregnancy and use of an electronic app can now help clinicians achieve this. These patients should ideally be managed by a multidisciplinary team whenever possible even remotely. Although clinical research has closed some knowledge gaps regarding the diagnosis and management of these women, there remains significant variation in practise and lack of evidence-based guidelines still exists in many spheres of clinical care in which caregivers must rely on expert opinion. Ongoing efforts in education and research will continue to improve care for these women and restore quality of life for them.

The role of integrin-mediated cell adhesion in atherosclerosis: pathophysiology and clinical opportunities.

Integrins have been reported to mediate cell survival, proliferation, differentiation, and migration programs. For this reason, the past few years have seen an increased interest in the implications of integrin receptors in atherosclerosis. This review considers the potential role of integrins in atherosclerosis and also addresses why integrins present attractive targets for drug design. It discusses the properties of the integrin-based chemotherapeutic agents currently under consideration clinically and endeavours to provide insights into development of cardiovascular drugs using integrins as targets.

Health-related quality of life in a cohort of adult patients with mild hemophilia A.

OBJECTIVES: To compare the health-related quality of life among adult males affected with mild hemophilia A due to the same mutation (Val2016ala) to that of unaffected age and sex matched controls from the same general population. METHODS: The Short-Form 36 (SF-36) and Health Assessment Questionnaire (HAQ) were used to measure health-related quality of life and physical function. Other measures included bleeding history, a measure of joint damage, body mass index, age, and viral infection status. Cross-sectional data were collected through research clinics and a retrospective chart audit over a two-year period. RESULTS AND CONCLUSIONS: The study included 47 affected males and 33 controls. The affected males had a higher level of co-morbidity, prior bleeding, and existing joint damage than controls. With the exception of the social function and health transition scales, mean scores for each of the SF-36 domains were worse among affected males. Mean differences were more than a clinically important five points in five of eight domains, with the general health scale showing more than a 10-point difference. Despite the degree of difference noted, only two of the differences were statistically significant (general health and role emotional scales) because of the small sample size and considerable individual variation in SF-36 scale scores. Multiple regression analyses suggested existing joint damage and presence of heart disease as the strongest associates of lower physical health-related quality of life. Joint damage in turn was partly related to prior hemarthroses. Compared to the Canadian population, affected males had lower scores in six out of eight SF-36 domains as well as the physical component summary score. There were no significant differences found in the HAQ scores between the two groups. So-called mild hemophilia A was associated with a negative effect on physical health-related quality of life, contributed to by joint damage as a result of prior bleeding.

Structure-activity relationship studies on ADAM protein-integrin interactions.

The ADAM (a disintegrin and metalloprotease) family of proteins possess multi-domain structures composed of a signal peptide, a prodomain, a metalloprotease domain, a disintegrin-like domain, a cysteine rich domain, an epidermal growth factor-like domain, a transmembrane domain and cytoplasmic tail. The disintegrin-like domain shares sequence similarity with the soluble venom disintegrins, a family of proteins which are potent inhibitors of integrin-mediated platelet aggregation and cell adhesion. Several ADAMs have been reported to interact with integrins, and the disintegrin-like domain may be crucial part in this respect. A description of structure-activity relationship of ADAM proteins interacting with integrin is outlined in this review. The review highlights recent reports on potential integrin family for ADAMs and how ADAMs selectively modulate interaction for integrin mediated cell function. Lastly, it describes progress in understanding the structural features and functional roles of the ADAMs in normal and pathological conditions and how this insight may assist the development of new therapeutic approaches.

Integrins in drug targeting-RGD templates in toxins.

Integrins are a family of heterodimeric receptors, which modulate many cellular processes including: growth, death (apoptosis), adhesion, migration, and invasion by activating several signaling pathways. Integrin-binding RGD (arginine-glycine-aspartic acid) is found in several important extracellular matrix proteins which serve as adhesive integrin ligands. The RGD motif has also been found in many toxins from snake venom and other sources that specifically inhibit integrin binding function and serve as potent integrin antagonists, particularly of platelet aggregation and integrin-mediated cell adhesion. Many of these proteins have potential as therapeutic agents which can target integrins directly. Structural and functional studies of several RGD-containing toxins suggest that the inhibitory potency of these proteins lies in subtle positional requirements of the tripeptide RGD at the tip of a flexible loop, a structural feature for binding to integrins. In addition, amino acid residues in this loop in close vicinity to the RGD-motif determine the integrin-binding specificity and selectivity. This review will present a review of integrin structure and function, and of disintegrin structural features responsible for their activity as antagonists of integrin function. The use of integrins in drug targeting and integrins as targets for drug delivery by using the RGD as a template structure will also be discussed.

Haemophilia and ageing.

Men with haemophilia have not only the challenges of living with HIV and/or HCV infection and premature arthritis as complications of their disorder, but they also confront the other ails of ageing. These include genitourinary problems such as prostatic hypertrophy, prostatic cancer and renal stone disease, and arterial disease for which haemophilia is not protective. Progressive arthritis and declining fitness may lead to loss of independence which causes great concern. Associated with the physical aspects of ageing, many patients also suffer from psychological symptoms which may be precipitated by changes in work such as early retirement and altered family dynamics. Many older men with haemophilia may never have consulted primary care physicians because of the rarity and complexity of their disorder. Haemophilia centre staff often assume responsibility for the identification and management of all health problems of their patients. Even when other clinicians are involved, patients require their centre's involvement in the investigation and support of many procedures such as coronary artery surgery and urological surgery. This paper addresses falls in the older man with haemophilia, their causes and consequences and cardiovascular problems in particular. Very little literature has been published about these common problems. We need to be aware of the ageing issues in haemophilia and develop 'wellness' programs which are directed to the early identification of disease as well as preventative strategies to reduce the physical and psychological impacts of ageing.

Treatment of intracranial and extracranial haemorrhages in a neonate with severe haemophilia B with recombinant factor IX infusion.

Intracranial (ICH) and extracranial (ECH) haemorrhages are potentially life-threatening events that may occur comorbidly in neonates with haemophilia. There is little data on the use of recombinant factor IX (rFIX; BeneFIX in the neonate. Children <15 years of age are known to require higher doses of recombinant Factor IX (FIX) than older persons, which raises specific concerns in the neonate due to the increased risk of thrombosis in this age group (Thromb Haemost 2002; 87: 431). This report describes a case in which a high rate of continuous infusion of recombinant FIX was used to treat a newborn with significant intracranial and subgaleal haemorrhages. A high rate of infusion maintained at 30-35 U kg(-1) h(-1) was necessary to maintain adequate FIX levels. Despite the high rate of continuous infusion, no adverse events were noted. Our patient had a rare genetic mutation causing severe haemophilia B. A neonate with severe haemophilia B was treated successfully with recombinant FIX through continuous infusion. A high rate of infusion was required and no complications were noted.

Snake venom metalloproteinase containing a disintegrin-like domain, its structure-activity relationships at interacting with integrins.

Snake venom disintegrins represent a family of RGD (Arg-Gly-Asp) or KGD (Lys-Gly-Asp)-containing proteins which have been reported to be unique and potentially useful tools not only for investigating integrin-ligand interactions, but also for the development of anti-thrombotic agents in terms of their anti-platelet activities. Snake venom proteins containing a disintegrin-like domain represent another super-family of proteins in which many of them have been demonstrated to have similar ability to inhibit platelet aggregation and integrin-mediated cell adhesion as the disintegrins. This super-family includes a large number of snake venom metalloproteinases and disintegrin related, RGD-containing snake venom proteins (disintegrin-like proteins) such as dendroaspin. Recently, a family of homologues of the snake venom metalloproteinases have been found in a wide variety of mammalian tissues as well as in other eukaryotic organisms termed ADAM (a disintegrin-like and metalloproteinase) proteins. ADAMs are members of the metazincins that also include the related matrix metalloprotease (MMPs). Some of ADAM proteins have now shown to interact with integrins, and the disintegrin-like domain may be crucial part in their function as proteases. A description of structure-activity relationships of snake venom proteins containing a disintegrin-like domain is outlined in this review, along with reports of the modulation of protein activity by recombinant mutation. Comparison is also made of the structural and functional features of the metalloproteinases in snakes compared with those from other species. The review is intended to provide insights in which may assist the development of new therapeutic approaches.

Retrospective audit of a continuous infusion protocol for haemophilia A at a single haemophilia treatment centre.

Continuous infusion (CI) of factor concentrates has been suggested to decrease the risk of bleeding and reduce cost in the treatment of bleeding disorders. Concerns have also been raised regarding stability and sterility of products administered by CI, the risk of local thrombophlebitis and an association with the development of an inhibitor in mild haemophilia. A retrospective chart review was conducted to investigate a CI protocol regarding product use, maintenance of FVIII levels and the frequency of complications including inhibitor development. Twelve patients with haemophilia A received recombinant factor VIII by CI a total of 18 times between April 1998 and September 2003. Ages ranged from 4 months to 75 years and indications for treatment included severe bleeds and surgical prophylaxis. The protocol which was audited required a bolus of 50 U kg(-1) of FVIII followed by CI at an initial rate of 4 U kg(-1) hr(-1). All infusions were administered by i.v. infusion after diluting the reconstituted concentrate in saline. There were no documented cases of significant bleeding, adverse reactions, thrombophlebitis or infection. Two mild haemophilia A patients developed a low titre inhibitor after receiving CI. It is not clear in either case that CI was the main contributing factor. Our CI protocol will now be modified to use less product, aiming for more cost-effectiveness.

Enhanced angiogenesis following allogeneic blood transfusion.

Blood transfusions are associated with recurrence of solid cancers. Angiogenesis is essential for cancer growth. Our aim was to determine for the first time in a prospective cohort study the effect of prestorage allogeneic leucodepleted SAGM (saline, adenine, glucose, mannitol) red cell transfusion on angiogenic factor levels and in vitro angiogenesis. Forty pretransfusion adult hospital inpatients were selected consecutively. Serum vascular endothelial growth factor (VEGF) and endostatin were measured in each patient before and after prestorage allogeneic leucodepleted SAGM red cell transfusion. All samples were exposed to an in vitro endothelial cell proliferation assay and 10 sample groups were also exposed to an in vitro whole angiogenesis assay. The median number of units transfused was 2 (minimum-maximum, 2-4). Twenty-nine (73%) patients had a rise in VEGF, with an overall increase of 118 pg/ml (quartiles -5, 306; P < 0.01). Twenty-eight (70%) patients had a decrease in endostatin, with an overall reduction of 1.2 ng/ml (quartiles 4.0, 0.0; P = 0.017). There was an overall 33% increase in endothelial cell proliferation (P < 0.01) and a 9.4% increase in in vitro whole assay angiogenesis (P < 0.01). Prestorage allogeneic leucodepleted SAGM red cell transfusions are associated with a favourable angiogenic factor imbalance and an elevation in in vitro angiogenesis.

Modulation of integrin-binding selectivity by mutation within the RGD-loop of snake venom proteins: a novel drug development approach.

Integrins are a family of heterodimeric class I transmembrane receptors, many of which bind to the RGD sequence in adhesive proteins and mediate the adhesive interactions of a variety of cells. The RGD motif has also been found in snake venom proteins that specifically inhibit integrin binding function and serve as potent integrin antagonists. The majority of these proteins interact with beta1 and beta3 associated integrins and their potency is at least 500-2000 times higher than short RGD peptides. Structural and functional studies suggest that the inhibitory potency of these proteins lies in subtle positional requirements of the tripeptide RGD that is harboured in a defined flexible loop. The integrin-binding specificity and selectivity of each of the proteins is controlled by amino acid residues in this loop in close vicinity to the RGD-motif. The review includes an overview of the structure and function of snake-venom integrin antagonists. The ability of these proteins to control platelet aggregation, cell adhesion and ligand binding is compared to that of short linear, cyclic RGD-peptides and RGD-containing proteins and the influence of modulation of amino acid residues flanking the RGD motif is also considered. The review is intended to provide insight into the development of novel inhibitors as drugs.

Platelets and cancer.

The presence of platelets in association with cancer deposits has been recognised for over 100 years; however, the recognition of a two-way interaction has been more recent. The link between cancer spread and platelet stimulation is pivotal to understanding of the hypercoagulable state found in most cancer patients. The assistance of platelets in cancer spread may provide opportunities to interrupt this relation, thus inhibiting metastasis.

Inhibition of human alpha-thrombin by a phosphonate tripeptide proceeds via a metastable pentacoordinated phosphorus intermediate.

X-ray crystallographic studies of human alpha-thrombin with a novel synthetic inhibitor, an acyl (alpha-aminoalkyl)phosphonate, reveal the existence of a pentacovalent phosphorus intermediate state. Crystal structures of the complex of alpha-thrombin with the phosphonate compound were determined independently using crystals of different ages. The first structure, solved from a crystal less than seven days old, showed a pentacoordinated phosphorus moiety. The second structure, determined from a crystal that was 12 weeks old, showed a tetracoordinated phosphorus moiety. In the first structure, a water molecule, made nucleophilic by coordination to His57 of alpha-thrombin, is bonded to the pentacoordinated phosphorus atom. Its position is approximately equivalent to that occupied by the water molecule responsible for hydrolytic deacylation during normal hydrolysis. The pentacoordinated phosphorus adduct collapses to give the expected pseudo tetrahedral complex, where the phosphorus atom is covalently bonded to Ser195 O(gamma). The crystallographic data presented here therefore suggest that the covalent bond formed between the inhibitor's phosphorus atom and O(gamma) of Ser195 proceeds via an addition-elimination mechanism, which involves the formation of a pentacoordinate intermediate.

High affinity binding of heparin by necrotic tumour cells neutralises anticoagulant activity--implications for cancer related thromboembolism and heparin therapy.

We have observed a striking neutralisation of the anticoagulant activity of unfractionated heparin in the presence of a pancreatic carcinoma cell line (MIA PaCa-2) due to binding of around 9 microg of heparin per 10(7) cells (apparent Kd, 30 nM). The loss of anticoagulant activity was less marked in the presence of low molecular weight forms of heparin. Binding to the cell blocked acceleration of the thrombin:antithrombin interaction by heparin. Neutralisation of heparin activity was also shown to occur in the presence of a number of other tumour cell lines. FACS analysis demonstrated that live cells did not bind heparin and high affinity binding only occurred to dead MIA PaCa-2 cells. Heparin binding proteins accumulating in cell medium were identified as histone and ribosomal proteins that will become exposed during necrosis. The release of these proteins from cells within the necrotic core of a tumour or from cells killed during chemotherapy may abrogate the heparan sulphate/antithrombin system and possibly contribute to the idiopathic thromboembolism often associated with cancer (Trousseau's syndrome). The findings also suggest a reason for the reported advantage of LMWH over UFH in treating venous thromboembolism in cancer patients and in improving patient survival.

Outcome of pediatric thromboembolic disease: a report from the Canadian Childhood Thrombophilia Registry.

The outcome for children with deep vein thrombosis (DVT) and pulmonary embolism (PE) is unknown. An understanding of morbidity and mortality of DVT/PE is crucial to the development of rational treatment protocols. The Canadian Childhood Thrombophilia Registry has followed 405 children aged 1 mo to 18 y with DVT/PE for a mean of 2.86 y (range, 2 wk to 6 y) to assess outcome. The all-cause mortality was 65 of 405 children (16%). Mortality directly attributable to DVT/PE occurred in nine children (2.2%), all of whom had central venous line-associated thrombosis. Morbidity was substantial, with 33 children (8.1%) having recurrent thrombosis, and 50 children (12.4%) having postphlebitic syndrome. Recurrent thrombosis and postphlebitic syndrome were more common in older children, although deaths occurred equally in all age groups. The incidence of recurrent thrombosis and postphlebitic syndrome are likely underestimated because of difficulties in diagnosis, especially in younger children. The significant mortality and morbidity found in our study supports the need for international multicenter randomized clinical trials to determine optimal prophylactic and therapeutic treatment for children with DVT/PE.

Bifunctional peptide boronate inhibitors of thrombin: crystallographic analysis of inhibition enhanced by linkage to an exosite 1 binding peptide.

The affinity of the hirudin49-64 segment for exosite 1 of thrombin has been used previously to enhance the potency of simple competitive inhibitors [DiMaio, J., Gibbs, B., Munn, D., Lefebvre, J. , Ni, F., Konishi, Y. (1990) J. Biol. Chem. 265, 21698-21703., and Maraganore, J. M., Bourdon, P., Jablonski, J., Ramachandran, K. L., and Fenton, J. W., II (1990) Biochemistry 29, 7095-7087.]. Using a similar approach, we have enhanced the activity of two active site directed thrombin inhibitors by attaching this segment via a novel reverse oriented linker to each of two tripeptide boronate inhibitors. At P1, compound 1 contains an arginine-like, isothiouronium, side chain, while compound 2 contains an uncharged, bromopropyl residue. Inhibition of human alpha-thrombin by compound 1 shows slow, tight-binding competitive kinetics (final Ki of 2.2 pM, k1 of 3.51 x 10(7) M-1 s-1, and k-1 of 1.81 x 10(-)4 s-1). The addition of hirugen peptide (20 microM) competes for exosite 1 binding and restores the k1 and k-1 to that of the analogous tripeptide, 0.29 x 10(7) M-1 s-1 and 0.13 x 10(-)4 s-1, respectively. Compound 1 has enhanced specificity for thrombin over trypsin with KiTry/KiThr of approximately 900 compared to the analogous tripeptide, with KiTry/KiThr of approximately 4. Compound 2 acts as a competitive inhibitor (KiThr of 0.6 nM) and is highly selective with no effect on trypsin. Crystallographic analysis of complexes of human alpha-thrombin with compound 1 (1.8 A) and compound 2 (1.85 A) shows a covalent bond between the boron of the inhibitor and Ser195 (bond lengths B-O of 1.55 and 1.61 A, respectively). The isothiouronium group of compound 1 forms bidentate interactions with Asp189. The P2 and P3 residues of the inhibitors form interactions with the S2 and S3 sites of thrombin similar to other D-Phe-Pro based inhibitors [Bode, W., Turk, D., and Karshikov, A. (1992) Protein Sci. 1, 426-471.]. The linker exits the active site cleft of thrombin forming no interactions, while the binding of Hir49-64 segment to exosite 1 is similar to that previously described for hirudin [Rydel, T. J., Tulinsky, A., and Bode, W. (1991) J. Mol. Biol. 221, 583-601.]. Because of the similarity of binding at each of these sites to that of the analogous peptides added alone, this approach may be used to improve the inhibitory activity of all types of active site directed thrombin inhibitors and may also be applicable to the design of inhibitors of other proteases.

Tissue factor pathway inhibitor in endothelial cells colocalizes with glycolipid microdomains/caveolae. Regulatory mechanism(s) of the anticoagulant properties of the endothelium.

Tissue factor pathway inhibitor (TFPI), the main downregulator of the procoagulant activity of tissue factor.factor VIIa complex, locates in human endothelial cells (EC) in culture as well-defined clusters uniformly distributed both on the cell surface and intracellularly. We here demonstrate by immunofluorescence that TFPI colocalizes in EC with caveolin, urokinase-type plasminogen activator receptor, and glycosphingolipids. The localization of TFPI in caveolae in resting endothelium is proved by double immunogold electron microscopy for TFPI and caveolin. After ultracentrifugation of rat lung or EC homogenates through density gradients of Nycodenz, TFPI was highly enriched at densities of 1.05 to 1.08 g/mL, together with caveolin and alkaline phosphatase. By ELISA, more than half of the cellular TFPI was detected in Triton X-100-insoluble extracts of EC. TFPI incorporates [1-3H]ethanolamine and is cleaved from the cell surface by phosphatidylinositol-phospholipase C, indicating a specific glycosylphosphatidylinositol-anchorage mechanism for TFPI in the plasma membrane. Clustering of TFPI and its localization in caveolae are dependent on the presence of cholesterol in the membrane. Agonist-induced stimulation of EC caused marked changes of distribution for both TFPI and caveolin at subcellular level, with subsequent increase of the cell surface-associated inhibitory activity toward tissue factor.factor VIIa. Our findings suggest that, beside their function in transcytosis, potocytosis, cell surface proteolysis, and regulation of signal transduction, caveolae also play a direct role in the regulation of EC anticoagulant properties.