The spectrum of bleeding in women and girls with haemophilia B.

Although hemophilia B affects 1 in 25,000 males there may be 3 female hemophilia B carriers per affected male. This clinical review highlights the unique challenges faced by hemophilia B carriers including the under-recognition of bleeding symptoms associated with and without FIX deficiency, discrepancies in correlation between genotype and bleeding phenotype and therapeutic considerations utilizing clinical vignettes of common scenarios.

Platelet thrombus formation in eHUS is prevented by anti-MBL2.

E. coli associated Hemolytic Uremic Syndrome (epidemic hemolytic uremic syndrome, eHUS) caused by Shiga toxin-producing bacteria is characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute kidney injury that cause acute renal failure in up to 65% of affected patients. We hypothesized that the mannose-binding lectin (MBL) pathway of complement activation plays an important role in human eHUS, as we previously demonstrated that injection of Shiga Toxin-2 (Stx-2) led to fibrin deposition in mouse glomeruli that was blocked by co-injection of the anti-MBL-2 antibody 3F8. However, the markers of platelet thrombosis in affected mouse glomeruli were not delineated. To investigate the effect of 3F8 on markers of platelet thrombosis, we used kidney sections from our mouse model (MBL-2+/+ Mbl-A/C-/-; MBL2 KI mouse). Mice in the control group received PBS, while mice in a second group received Stx-2, and those in a third group received 3F8 and Stx-2. Using double immunofluorescence (IF) followed by digital image analysis, kidney sections were stained for fibrin(ogen) and CD41 (marker for platelets), von-Willebrand factor (marker for endothelial cells and platelets), and podocin (marker for podocytes). Electron microscopy (EM) was performed on ultrathin sections from mice and human with HUS. Injection of Stx-2 resulted in an increase of both fibrin and platelets in glomeruli, while administration of 3F8 with Stx-2 reduced both platelet and fibrin to control levels. EM studies confirmed that CD41-positive objects observed by IF were platelets. The increases in platelet number and fibrin levels by injection of Stx-2 are consistent with the generation of platelet-fibrin thrombi that were prevented by 3F8.

Shiga toxin enhances functional tissue factor on human glomerular endothelial cells: implications for the pathophysiology of hemolytic uremic syndrome.

BACKGROUND: The pathogenesis of Shiga toxin (Stx)-mediated childhood hemolytic uremic syndrome (HUS) is not fully delineated, although current evidence implicates a prothrombotic state. We hypothesized that the tissue factor (TF) pathway plays a major role in the pathophysiology of HUS. MATERIALS AND METHODS: We measured cell surface TF activity in response to tumor necrosis factor-alpha (TNF-alpha) (20 ng mL(-1), 2-144 h), Stx-1 (10(-11) mol L(-1), 4-144 h), or their combination (TNF-alpha 22 h and Stx-1 for the last 0.5-4 h of TNF-alpha incubation) on human glomerular (microvascular) endothelial cells (HGECs) and human umbilical vein (macrovascular) endothelial cells (HUVECs). RESULTS AND CONCLUSIONS: We observed that while TNF-alpha caused an increase in cell surface TF activity on both cell types, the combination of TNF-alpha and Stx-1 differentially affected HGECs. On these cells, TF activity was increased further by 2.67 +/- 0.38-fold (n = 38, P < 0.001), consistent with our parallel observation that Stx-1 binds to HGECs but not to HUVECs. Anti-TF antibody abolished functional TF while anti-tissue factor pathway inhibitor antibody enhanced TF activity. Stx-1 alone did not induce TF activity on either cell type. Measurement of TF antigen levels and quantitative real-time polymerase chain reaction demonstrated that exposure to TNF-alpha markedly increased TF protein and TF mRNA for HGECs, but the exposure to the combination of TNF-alpha and Stx-1 did not increase further the amount of either TF protein or TF mRNA. We conclude that cytokine-activated HGECs, but not HUVECs, undergo a significant augmentation of cell surface TF activity following exposure to Stx, suggesting an important role for TF in the coagulopathy observed in HUS.

Shear stress decreases endothelial cell tissue factor activity by augmenting secretion of tissue factor pathway inhibitor.

Monolayers of human umbilical vein endothelial cells were activated with 50 U/mL interleukin-1alpha (IL-1alpha) for 3 hours and simultaneously conditioned with shear stresses of 0, 0.68, or 13.2 dyne/cm(2) in a parallel-plate flow chamber. In the presence of an inflow buffer containing 100 nmol/L factor X and 10 nmol/L factor VII, production of factor Xa, a measure of functional tissue factor (TF), was determined as the product of outflow concentration of factor Xa (chromogenic assay performed under quasi-static flow conditions after the shear period) and flow rate. Similarly, production of TF pathway inhibitor (TFPI) was estimated as the product of antigenic TFPI (by enzyme-linked immunosorbent assay) in the supernatant and flow rate. In parallel experiments, total RNA was isolated for determination of amplification products of TF mRNA by reverse transcription-polymerase chain reaction. We found that shear stress reduced factor Xa production (mean+/-SE; n=number of experiments) from 13.33+/-1.14 (n=16) fmol/minxcm(2) at 0 shear stress to 5.70+/-2.51 (n=5) and 0.54+/-0.54 (n=4) fmol/minxcm(2) at shear stresses of 0.68 and 13.2 dyne/cm(2), respectively. At the same time, immunogold labeling showed that TF antigen on the endothelial surface increased >5-fold with shear stress, whereas TFPI antigen on the surface increased 2-fold. The secretion of TFPI (appearance of new supernatant TFPI) rose from 7.4+/-2.4 (n=12) x10(-)(3) fmol/minxcm(2) at 0 shear stress to 23.7+/-7.3 (n=9) and 50.2+/-14.3 (n=4) x10(-)(3) fmol/minxcm(2) at 0.68 and 13.2 dyne/cm(2), respectively. TF mRNA amplification products were not markedly changed by shear stress. We conclude that acute application of shear stress reduces functional, but not antigenic, expression of TF by intact, activated endothelial cell monolayers in a manner associated with shear stress-augmented endothelial cell secretion of TFPI.

Endothelial cell function, including tissue factor expression, under flow conditions.

Vascular endothelium remains a dynamic interface between blood and the vessel wall. New developments make clear that many antithrombotic and prothrombotic responses of the endothelium depend on flow conditions in an adaptive manner: duration of a certain level of shear stress matters as well as level of shear. In general, over a time course of several hours, endothelium appears to be more actively antithrombotic under moderate shear conditions (e.g., 15 dynes/cm2), and more fibrinolytic under high (e.g., 30 dynes/cm2). Pulsatile flow and cyclic wall stress further modify these responses. Special consideration, moreover, must be given to branch points and regions of irregular geometry (i.e., stenoses, aneurysms) in the circulation. In such locations, predilection sites for thrombosis, lipid uptake, and atherosclerosis, low levels of fluid shear stress (e.g., 0.5 dynes/cm2), large gradients in fluid shear stress, and vessel wall bending stresses all become important. Preliminary work suggests that endothelial cells in such regions can become prothrombotic, leading to localized platelet adhesion/aggregation and fibrin formation on subendothelium and perhaps deeper structures following vessel injury.

A hematologist's view of contrast media, clotting in angiography syringes and thrombosis during coronary angiography.

While ionic contrast media (CM) are stronger anticoagulants and antiplatelet agents, both nonionic and ionic CM retard clotting, fibrinopeptide A generation and platelet aggregation (at least by Born-O'Brien aggregometry). Thus, nonionic CM do not cause clots and thrombi. Rather, the driving force for clot or thrombus formation, when it occurs, is blood contact with and activation by the foreign surface of a syringe or catheter itself. A marked enhancement of clotting by glass syringes in comparison to plastic ones supports this view. Blood in any syringe or catheter, therefore, will clot more slowly in the presence of nonionic or ionic CM, the inhibitory effects of the latter being more profound. With respect to models of thrombosis at sites of vascular injury or stenosis, the antithrombotic effects of CM may either be transient owing to the dynamic nature of blood flow (local endothelial cell denudation model), or as in the case of ionic CM, actually to enhance local platelet aggregation (stenosis model). In these situations, preservation of the antithrombotic functions of endothelium with nonionic CM may be quite critical.

Effects of contrast media on endothelial cell monolayers under controlled flow conditions.

To compare the biocompatibility of nonionic and ionic intravascular contrast media in a more physiologic in vitro system, key aspects of blood flow through a microvessel were simulated. Toward this end, monolayers of endothelial cells placed in a specially designed flow chamber, real-time imaging of the monolayers by brightfield and phase videomicroscopy, and real-time imaging and computer-aided quantitation at normal hematocrit levels of platelet adhesion/aggregation to sites of monolayer injury by means of epifluorescence videomicroscopy were used. At a concentration in culture medium of 20% by volume, it was found that monolayer morphology was least altered by iohexol when compared with diatrizoate and ioxaglate; monolayer production of prostacyclin was enhanced (p less than 0.001) by ioxaglate compared with saline controls; and at a concentration in citrated blood of 20% by non-red-cell volume, platelet adhesion/aggregation was reduced by all 3 contrast agents in the order diatrizoate greater than ioxaglate greater than iohexol.

Effects of contrast media on erythrocyte and platelet interactions with endothelial cell monolayers exposed to flowing blood.

Although there have been reports of clot formation in angiographic syringes containing nonionic contrast agents, these clots are now believed to be aggregates of red cells that form in the low ionic strength, low pH, zero shear rate environment of contrast media syringes. In an in vitro controlled flow system that stimulates blood flow through a microvessel, we found that large (100 microns or more) red cell aggregates are not seen with any of the media tested (iohexol, sodium methylglucamine diatrizoate, and ioxaglate); aggregates that form under shear conditions are rouleaux. The degree of rouleau formation, compared with saline controls, is greatest with ioxaglate, least with diatrizoate, and intermediate with iohexol. Platelet adhesion/aggregation at a site of injury to vascular endothelium is not significantly affected by iohexol. Rouleaux formation, a potential determinant of local levels of thrombin and other platelet-activating substances, is shown to occur in the lee of mural platelet thrombi.

Nonionic contrast media. Procoagulants or clotting innocents?

OBJECTIVES: Compared with ionic contrast media, nonionic contrast media cause fewer adverse reactions, including hemodynamic and electrophysiologic complications, and are better tolerated by patients. However, concern as to whether nonionic agents are associated with more thromboembolic complications has been raised in recent years. This article reviews in-vitro and in-vivo coagulation studies with nonionic contrast media, including the authors' current study using blood samples from percutaneous transluminal coronary angioplasty (PTCA) patients. METHODS: Articles for this review were selected on the basis of providing viewpoints representative of both sides of the clotting controversy involving nonionic contrast media. In addition, articles were selected that, in toto, include the several relevant aspects of thrombosis during PTCA: whole blood clotting, angiography catheters, angiography syringes, rheology, and vascular endothelium. The preliminary work described used platelet adhesion/aggregation to a collagen-coated surface under controlled conditions of heparinized, whole blood flow. Adhesion/aggregation was quantified by digital analysis of real-time images obtained by epifluorescence videomicroscopy. In parallel studies, changes in markers (membrane glycoproteins) of platelet activation were measured using whole blood flow cytometry. RESULTS: Although nonionic contrast media are weaker anticoagulants than ionic contrast media, no convincing evidence demonstrates that they are procoagulant. On the contrary, in-vitro studies suggest that ionic media may weaken the antithrombotic properties of vascular endothelium by direct endothelial injury. Certain clinical trials of PTCA patients substantially challenge the use of nonionics. Yet, Grabowski et al found no evidence of platelet activation by ioxaglate or iohexol in preliminary work. This result was obtained with two of the most sensitive platelet assays available: flowing whole blood aggregometry and whole blood flow cytometry. Attention should be given to the adequacy of heparinization in future clinical studies. CONCLUSIONS: Nonionic contrast media are not procoagulant in vitro in the hands of most research groups. The focus of current controversy, therefore, is whether there is a clinical and in-vivo difference in thrombotic events seen with nonionic versus ionic media during PTCA. Elements in this controversy include platelet activation, blood-foreign surface (catheter and syringe) interaction, rheology, vascular endothelium, and adequacy of heparinization.

Anticoagulant effects of nonionic versus ionic contrast media in angiography syringes.

To determine whether nonionic contrast media present a clotting hazard when plastic or glass injection syringes are contaminated with aspirated blood, we evaluated two nonionic (iohexol and iopamidol) and two ionic (ioxaglate and diatrizoate) contrast agents. We used a blood:contrast media ratio of 2 mL:5 mL and ten normal donors, each studied at 10, 20, 30, and 60 minutes, a parallel study of clotting and fibrinopeptide A (FPA) generation in plastic tubes, and life table analysis to estimate more accurately donor-based early clotting probabilities. While ionic contrast media are stronger anticoagulants, both nonionic and ionic media retard clotting in plastic tubes, and clotting in plastic and glass angiography syringes in comparison to saline controls. A clotting probability of 1% for nonionic agents in plastic syringes was not reached until a time (mean +/- SD) of 21.5 +/- 3.2 minutes. This contrasts with a time of 8.7 +/- 2.5 minutes for saline control. With plastic syringes, no clotting at all was observed at 10 and 20 minutes with either class of agents. Neither class of agents hastened the generation of FPA. We found no evidence, therefore, that nonionic agents either cause clots or are procoagulant.

Endothelial cell modulation of primary platelet hemostasis.

Rheology has profound effects on the rate, structure, and modulation of primary hemostasis. Many of these effects can be studied via real-time, epifluorescence videomicroscopy of platelet adhesion-aggregation to a site of injury to an endothelial cell monolayer exposed to flowing blood. In particular, with the model described, endothelial cells can be shown to be a significant modulator of platelet function at an injury site.

Prostacyclin production by internal mammary artery as a factor in coronary artery bypass grafts.

Long-term patency of coronary artery bypass grafts (CABG) with internal mammary artery (IMA) is better than with saphenous vein (SV) grafts. To determine if vascular prostacyclin (PGI2) produced by IMA might contribute to the improved outcome, we compared PGI2 generated by IMA and SV fragments from 26 patients undergoing CABG and tested the effect of preoperative, long-term ingestion of of aspirin. Fresh tissues were incubated in buffer +/- 25 mumol/L of sodium arachidonate at 37 degrees C for 5 minutes to stimulate PGI2 production, measured by radioimmunoassay of its major hydrolytic product, 6-keto-PGF1 alpha. Results were expressed in picograms of 6-keto-PGF1 alpha per milligram tissue wet weight for total PGI2 production by vascular segments and picograms per cm2 surface area for endothelial PGI2 production. Endothelial PGI2 production was compared for IMA and SV in template-stirring chambers that exposed only the luminal surface of the vessel, excluding underlying smooth muscle. Endothelial PGI2 production by IMA was significantly higher than production by SV under both basal (mechanical stimulation only 1436 +/- 224 versus 842 +/- 227 pg/cm2, mean +/- SEM, p greater than 0.05) and stimulated (25 mumol/L sodium arachidonate: 3343 +/- 347 versus 2032 +/- 465 pg/cm2, p less than 0.025) conditions in patients not receiving aspirin. For patients receiving aspirin, endothelial PGI2 production by IMA was significantly higher than production by SV in stimulated conditions (1382 +/- 526 versus 683 +/- 124 pg/cm2, p less than 0.05). Histologic examination of the tissue segments revealed intact endothelium after incubation in both IMA and SV. Thus a high capacity for PGI2 synthesis and diminished inhibition of PGI2 after aspirin were demonstrated for IMA compared with SV tissue and may be a factor in the improved patency of IMA grafts.

Intraocular and extraocular retinoblastoma.

Retinoblastoma, the most common primary ocular malignancy of childhood, is a tumor in which the pediatrician and pediatric oncologist can now play a much more significant role in therapy. Developments in molecular biology have now made carrier testing and prenatal diagnosis feasible. In the near future, these developments should greatly augment the pediatrician's and pediatric oncologist's ability to offer accurate and appropriate genetic counseling for affected families. A practical staging system for extraocular retinoblastoma together with stage-related effective chemotherapy and radiation therapy was presented in this chapter. These modalities now make possible long-term survival for the majority of the 1 out of 8 children with retinoblastoma who would otherwise die from metastatic disease. Finally, 40 per cent of all children with retinoblastoma (those with the germinal mutation) are at lifelong risk for second, nonocular malignancies. The recognition that more than half of these children will actually develop second tumors by the fourth decade of life makes vigilant follow-up care for these patients a necessity.

Variability of platelet degranulation by different contrast media.

RATIONALE AND OBJECTIVES: It has been suggested that nonionic but not ionic contrast media degranulate blood platelets when mixtures of blood and contrast media are studied by flow cytometry. This phenomenon was further assessed in the current study not only by performing whole-blood platelet flow cytometry but also by performing flowing blood platelet aggregometry. The latter is a highly sensitive measure of platelet function. METHODS: Blood samples were collected from six normal donors and mixed with equal volumes of an ionic monomer (diatrizoate), a nonionic monomer (iohexol), an ionic dimer (ioxaglate), and a nonionic dimer (iodixanol). Samples were collected in the presence of no anticoagulant for 1 min prior to the addition of sodium citrate or in the presence of heparin (14.5 U/ml) or recombinant hirudin (60 micrograms/ml). All samples were fixed in formaldehyde within 30 min. RESULTS: Platelet degranulation was observed with one nonionic agent (iohexol) and one ionic agent (diatrizoate). Degranulation was not seen with iodixanol or ioxaglate. CONCLUSION: These findings indicate that degranulation is independent of the ionic or nonionic nature per se of contrast media. A possible explanation for this conclusion is suggested.