von Willebrand disease (VWD): evidence-based diagnosis and management guidelines, the National Heart, Lung, and Blood Institute (NHLBI) Expert Panel report (USA).

von Willebrand disease (VWD) is a commonly encountered inherited bleeding disorder affecting both males and females, causing mucous membrane and skin bleeding symptoms, and bleeding with surgical or other haemostatic challenges. VWD may be disproportionately symptomatic in women of child-bearing age. It may also occur less frequently as an acquired disorder (acquired von Willebrand syndrome). VWD is caused by deficiency or dysfunction of von Willebrand factor (VWF), a plasma protein that mediates platelet haemostatic function and stabilizes blood coagulation factor VIII. The pathophysiology, classification, diagnosis and management of VWD are relatively complex, but understanding them is important for proper diagnosis and management of patients with VWD. These evidence-based guidelines for diagnosis and management of VWD from the National Heart, Lung, and Blood Institute (NHLBI) Expert Panel (USA) review relevant publications, summarize current understanding of VWD pathophysiology and classification, and present consensus diagnostic and management recommendations based on analysis of the literature and expert opinion. They also suggest an approach for clinical and laboratory evaluation of individuals with bleeding symptoms, history of bleeding or conditions associated with increased bleeding risk. This document summarizes needs for further research in VWF, VWD and bleeding disorders, including clinical research to obtain more objective information about bleeding symptoms, advancements in diagnostic and therapeutic tools, and enhancement in the education and training of clinicians and scientists in bleeding and thrombotic disorders. The NHLBI Web site (http://www.nhlbi.nih.gov/guidelines/vwd) has a more detailed document, a synopsis of these recommendations, and patient education information.

The effects of epinephrine infusion in patients with von Willebrand's disease.

Epinephrine infusion causes variable increases in the components of the Factor VIII (antihemophilic factor) complex in patients with von Willebrand's disease. The increase in antihemophilic factor procoagulant activity was greater than that of Factor VIII-related antigen and von Willebrand factor activity in two patients with von Willebrand's disease. Similar increases in the three individual factors were demonstrated in two other patients. A 4-10-fold increase in Factor VIII-related properties was identified in each of these individuals after infusion. One patient has been studied with very severe von Willebrand's disease; none of the Factor VIII-related properties increased despite two infusions of epinephrine. Bleeding times were normalized or remained normal in the two patients whose von Willebrand factor activity was greater than 25 U/100 ml. It remained prolonged in those three patients whose von Willebrand factor activity levels remained below that concentration. The increase in procoagulant activity was transient in all patients and t 1/2 values were estimated to be between 0.8 and 3.4 h.

Von Willebrand's disease with spontaneous platelet aggregation induced by an abnormal plasma von Willebrand factor.

We have investigated and characterized the abnormalities in four unrelated patients with von Willebrand's disease (vWd) who have (a) enhanced ristocetin-induced platelet aggregation (RIPA) at low ristocetin concentrations, (b) absence of the largest plasma von Willebrand factor (vWf) multimers, and (c) thrombocytopenia. The platelet-rich plasma of these patients aggregates spontaneously without the addition of any agonists. When isolated normal platelets are resuspended in patient plasma spontaneous aggregation occurs; however, the patients' plasmas did not induce platelet aggregation of normal washed formalinized platelets. When the patients' platelets are suspended in normal plasma, spontaneous aggregation is not observed. The spontaneous platelet aggregation (SPA) is associated with dense granule secretion as measured by ATP release and alpha granule release as measured by beta-thromboglobulin and platelet factor 4 release. The SPA is totally inhibited by 5 mM EDTA, prostaglandin I2, and dibutryl cyclic AMP, while it is only partially inhibited by 1 mM EDTA, acetylsalicylic acid, or apyrase. A monoclonal antibody directed against glycoprotein Ib (GPIb) and/or a monoclonal antibody against the glycoprotein IIb/IIIa (GPIIb/IIIa) complex totally inhibits the SPA. The vWf was isolated from the plasma of one of these patients. The purified vWf induced platelet aggregation of normal platelets resuspended in either normal or severe vWd plasma, but the vWf did not induce platelet aggregation of normal platelets resuspended in afibrinognemic plasma. Sialic acid and galactose quantification of the patient's vWf revealed approximately a 50% reduction compared with normal vWf. These studies indicate that a form of vWd exists, which is characterized by SPA that is induced by the abnormal plasma vWf. The SPA is dependent on the presence of plasma fibrinogen, and the availability of the GPIb and the GPIIb/IIIa complex. In this variant form of vWd the abnormal vWf causes enhanced RIPA, SPA, and thrombocytopenia.

Studies on a circulating anticoagulant in procainamide-induced lupus erythematosus.

Circulating anticoagulant activity that had at least two distinct mechanisms--one directed against factor XII and one directed against blood thromboplastin (prothrombin activator complex)--developed in a patient with clinical and laboratory evidence of procainamide hydrochloride-induced systemic lupus erythematosus. The anticoagulant activity behaved as a gamma-globulin in chromatographic and electrophoretic analyses, with the majority of activity behaving as an IgM immunoglobulin. Despite markedly abnormal coagulation study results, no clinical bleeding occurred. Anticoagulant activity paralleled clinical and laboratory evidence of the inflammatory disease and improved on discontinuance of procainamide therapy.

Toxicity of a first-generation adenoviral vector in rhesus macaques.

We constructed a first-generation adenovirus vector (AVC3FIX5) that we used to assess the rhesus macaque as a nonhuman primate model for preclinical testing of hemophilia B gene therapy vectors. Although we succeeded in our primary objective of demonstrating expression of human factor IX we encountered numerous toxic side effects that proved to be dose limiting. Following intravenous administration of AVC3FIX5 at doses of 3.4 x 10(11) vector particles/kg to 3.8 x 10(12) vector particles/kg, the animals in our study developed antibodies against human factor IX, and dose-dependent elevations of enzymes specific for liver, muscle, and lung injury. In addition, these animals showed dose-dependent prolongation of clotting times as well as acute, dose-dependent decreases in platelet counts and concomitant elevation of fibrinogen and von Willebrand factor. These abnormalities may be caused by the direct toxic effects of the adenovirus vector itself, or may result indirectly from the accompanying acute inflammatory response marked by elevations in IL-6, a key regulator of the acute inflammatory response. The rhesus macaque may be a useful animal model in which to evaluate mechanisms of adenovirus toxicities that have been encountered during clinical gene therapy trials.

Measurement of von Willebrand factor cleaving protease (ADAMTS-13): results of an international collaborative study involving 11 methods testing the same set of coded plasmas.

BACKGROUND: ADAMTS-13 is a von Willebrand factor (VFW)-cleaving protease. Its congenital or acquired deficiency is associated with thrombotic thrombocytopenic purpura (TTP) and more rarely with the hemolytic uremic syndrome. We report on a survey evaluating 11 methods for ADAMTS-13 measurement performed in different labs. DESIGN: Two plasmas, one normal and one from a patient with familial TTP, were mixed at the co-ordinating center to prepare 6 plasmas with 0%, 10%, 20%, 40%, 80% and 100% ADAMTS-13 levels. Each plasma was aliquoted and assembled into sets of 60 (coded from 1 to 60), each containing 10 copies of the original 6 plasmas. Plasmas were frozen and shipped in dry ice to 10 labs with a common frozen reference plasma. Laboratories were asked to measure ADAMTS-13 with their methods. Results were sent to the coordinating center for statistical analysis. RESULTS: Of the 10 methods performed under static conditions 9 were quantitative and one was semiquantitative. One method performed under flow conditions evaluated the extent of cleavage of endothelial cell-derived ultralarge VWF string-like structures and expressed results as deficient, normal, or borderline. Linearity (expected-vs-observed levels), assessed as the squared correlation coefficient, ranged from 0.98 to 0.39. Reproducibility, expressed as the coefficient of variation for repeated measurements, ranged from < 10% to 83%. The majority of methods were able to discriminate between different ADAMTS-13 levels. The majority were able to detect the plasma with 0% level and some of them to discriminate between 0% and 10%. Overall the best performance was observed for three methods measuring cleaved VWF by ristocetin cofactor, collagen binding, and immunoblotting of degraded multimers of VWF substrate, respectively. The poor interlaboratory agreement of results was hardly affected by the use of the common standard. The method performed under flow conditions identified the plasmas with 0%, 10%, 20% and 40% activity as deficient in 7, 5, 1 and 3 of the 10 replicate measurements. The plasmas with 80% and 100% were identified as normal in all of the 10 replicate measurements. CONCLUSIONS: The survey shows varied performance, but supports an optimistic view about the reliability of current methods for ADAMTS-13.

Pathogenesis of hypercalcemia in lymphosarcoma cell leukemia. Role of an osteoclast activating factor-like substance and a mechanism of action for glucocorticoid therapy.

The pathogenesis of hypercalcemia and mode of action of glucocorticoid therapy was examined in a patient with lymphosarcoma cell leukemia. Circulating neoplastic cells were cultured in vitro and secreted a bone-resorbing factor. The bone-resorbing factor was partially purified with the use of a bioassay for bone resorption, and was found to be chromatographically and pharmacologically similar to osteoclast activiating factor (OAF), which is produced by normal mitogen-activated peripheral blood lymphocytes. Other factors which stimulate bone resorption, such as parathyroid hormone, prostaglandins and the vitamin D metabolites, were excluded by criteria which included dose-response curves, radioimmunoassays, extraction in organic solvents and failure of glucocorticoids to inhibit bone-resorbing activity. The patient's hypercalcemia responded rapidly to prednisone therapy. The effects of the bone-resorbing factor secreted by the neoplastic cells on bone cultures to which cortisol was added were examined. Cortisol inhibited bone resorption directly at low doses (10(-8) M), which suggests that prednisone may have lowered the serum calcium in this patient by direct inhibition of bone resorption.

Characterization of a monoclonal antibody, OVB1, which binds to a unique determinant in human ovarian carcinomas and myeloid cells.

A monoclonal antibody, OVB1, was generated against a human ovarian carcinoma cell line, OVCAR-3. The antigen reacting with this antibody was strongly expressed on the external surface of the plasma membrane of OVCAR-3 cells and cells of 4/4 other ovarian carcinoma lines. Variable density and homogeneity of expression was found on cells from 5/5 breast carcinoma lines. Various ovarian tumor specimens and normal human tissues were frozen, cryostat-sectioned, and examined for OVB1 reactivity using immunoperoxidase methods. A strong, uniform, homogeneous reaction on 10/10 ovarian carcinoma specimens and variable, non-homogeneous reactions on breast tumors were seen. Normal tissues reacting with the antibody include thyroid, pituitary pars intermedia, breast ductal epithelium, Auerbach's plexus and neuronal processes in the GI tract, colonic mucosal epithelium, and salivary gland ductal epithelium. Polymorphonuclear leukocytes, eosinophils, and approximately 13% of peripheral lymphocytes, as well as cells around germinal centers in lymph nodes and spleen, showed strong reactivity by immunofluorescence and/or immunoperoxidase. Expression of the OVB1 antigen in the myeloid cells of normal human bone marrow occurred from the promyelocyte stage through to more mature cells in a subpopulation of myeloblasts. Indirect immunofluorescence of live peripheral blood cells showed localization to the surface of PMNs, eosinophils, and certain lymphocytes. Double-immunofluorescence studies (with a direct fluorescein-anti-lactoferrin antibody conjugate) showed co-localization of OVB1 and OKM1 (anti-C3bi receptor) antibodies to specific granules of PMNs. Localization of OVB1 and OKM1 antibodies to granular structures in the PMN was confirmed by electron microscopy using the ferritin bridge technique. The antigen reacting with the OVB1 antibody was shown to be neuraminidase sensitive, but protease insensitive. The OVB1 monoclonal antibody may be useful in identification of ovarian tumors and subclassification of myeloid leukemias.

Plasma fibrinopeptide A, beta-thromboglobulin, and platelet factor 4 in diabetic retinopathy.

We examined plasma levels of fibrinopeptide A, beta-thromboglobulin, and platelet factor 4 in diabetic patients. Among diabetic patients (n = 33) plasma levels of fibrinopeptide A, beta-thromboglobulin and platelet factor 4 were significantly higher than in controls (n = 41). In the subgroups of diabetic patients with (1) minimal (n = 13), and (2) moderate-severe (n = 14) retinopathy only plasma fibrinopeptide A levels were significantly higher than in controls. Among the total group of diabetic patients plasma levels of fibrinopeptide A increased significantly with increasing severity of retinopathy. These results suggest that diabetic retinopathy is associated with in vivo activation of blood coagulation factors and that this activation increases with advancing retinopathy.

Bleeding disorders in pregnant patients with rheumatic diseases.

Although the bleeding disorders that occur in patients with rheumatic diseases are not different during pregnancy than at other times, their diagnosis and management are often different during pregnancy. In idiopathic thrombocytopenic purpura, for instance, antiplatelet antibodies may cross the placenta and cause life-threatening thrombocytopenia in the fetus while the mother is asymptomatic. Management of this disease has changed significantly in the past 5 years with the use of intravenous gammaglobulin which appears to lessen the degree of fetal as well as maternal thrombocytopenia when administered during the peripartum period. The utilization of plasmapheresis and plasma infusions for patients with thrombotic thrombocytopenic purpura has salvaged both the mother and the fetus in a disease which was fatal in more than 60 per cent of patients prior to their use. The outcome of pregnancy in this patient population has markedly improved with this treatment. The stimulus for the production of factor VIII inhibitors in the postpartum period is still not understood, but guidelines for management have changed, with the increased likelihood of decreasing the antibody and inducing tolerance with regimens including factor VIII, immunosuppressive agents and intravenous gammaglobulin in those patients who require treatment. The diagnosis of von Willebrand's disease during pregnancy is difficult because of the physiologic increase in von Willebrand factor during pregnancy; in this instance family studies may help in the diagnosis of this relatively common, autosomal dominant inherited disorder. Management now includes treatment with desmopressin as well as cryoprecipitate replacement therapy.

Implications of immunologic methods for measuring antihemophilic factor (factor VIII).

Recent immunologic studies have provided important new information about the nature of AHF deficiency diseases and the structure and synthesis of this protein. They provide, moreover, a considerably improved ability to identify the carrier state in hemophilia A. Additional progress may be possible in the identification of carriers and of hemophilic patients at risk of antibody formation, but it will require a different approach to these problems. While preliminary goals have been obtained by using antibodies that identify antigenic determinants shared by normal and nonfunctional AHF, further progress will require the capacity to identify differences between the normal protein and the nonfunctional AHF-like molecules in hemophilic plasma.

A rapid method to visualize von willebrand factor multimers by using agarose gel electrophoresis, immunolocalization and luminographic detection.

A highly sensitive and rapid clinical method for the visualization of the multimeric structure of von Willebrand Factor in plasma and platelets is described. The method utilizes submerged horizontal agarose gel electrophoresis, followed by transfer of the von Willebrand Factor onto a polyvinylidine fluoride membrane, and immunolocalization and luminographic visualization of the von Willebrand Factor multimeric pattern. This method distinguishes type 1 from types 2A and 2B von Willebrand disease, allowing timely evaluation and classification of von Willebrand Factor in patient plasma. It also allows visualization of the unusually high molecular weight multimers present in platelets. There are several major advantages to this method including rapid processing, simplicity of gel preparation, high sensitivity to low concentrations of von Willebrand Factor, and elimination of radioactivity.

The identification and functional significance of factor VIII components on normal platelets.

Factor VIII-related antigen (VIII R:Ag) has previously been identified on human platelets by fluorescein-labelled heterologous antibodies to factor VIII. However, it is not known whether these antibodies identify only VIII R:Ag or whether antigens associated with factor VIII coagulant activity (VIII:C Ag) are also present. The current studies utilize specific antibodies and their Fab' fragments to evaluate the presence of both VIII R:Ag and VIII:C Ag on human platelets and to study the functional role of the platelet-associated VIII R:Ag on ristocetin-induced platelet agglutination. Immunofluorescent studies demonstrate the presence of VIII R:Ag, but do not identify VIII:C Ag on platelets. The functional studies reveal that after incubation with either of the Fab' fragments, platelets retain their reactivity in the ristocetin cofactor assay. These results indicate that this population of VIII R:Ag is not essential for the ristocetin aggregation reaction and that no antigenic determinants for factor VIII coagulant activity (VIII:C) are detectable on human platelets by immunofluorescence.

Platelet von Willebrand factor: comparison with plasma von Willebrand factor.

Platelet von Willebrand factor (vWf) was compared to its plasma counterpart. The platelet vWf was different from plasma vWf in that the multimeric organization was different, larger multimers were present, and the ratio of vWf activity to antigen was higher. Platelet and plasma vWf were similar in their antigenic reactivity in the electroimmunoassay and by liquid phase radioimmunoassay. The amount of vWf activity in large platelets was significantly higher than in normal platelets while the antigen content, although somewhat higher, was not significant. These studies show differences between normal platelet and plasma vWf, and also suggest that platelet size must be considered when platelet vWf is measured in disease states.

Diagnosis and management of von Willebrand's syndrome.

von Willebrand's disease is the most common of the inherited bleeding disorders. It is caused by quantitative and/or qualitative abnormalities of von Willebrand factor, and it usually presents with bleeding from mucosal surfaces. The diagnosis is confirmed by measuring von Willebrand factor activity and antigen levels, factor VIII activity, and performing a multimer analysis of von Willebrand factor. Treatment may require plasma-derived concentrates, but can often be accomplished with DDAVP, a vasopressin analogue that causes transient release of von Willebrand factor from body storage sites.

Retinal cotton-wool spots: an early finding in diabetic retinopathy?

Five insulin dependent diabetic patients are reported on who had a few small retinal cotton-wool spots or 'soft exudates' either totally isolated or associated with fewer than 10 microaneurysms. These observations suggest that cotton-wool spots may be an early finding in diabetic retinopathy. Significant biological abnormalities in these patients were high levels of glycosylated haemoglobin and mild increases in thrombin generation, indicating slight activation of the coagulation system. The possible significance of these clinical and biological findings is discussed.

Laboratory diagnosis of von Willebrand's disease.

The diagnosis of von Willebrand's disease is becoming complex as more is understood about the disease. Clinical information and laboratory data are necessary for the diagnosis because of the overlap of normal and abnormal laboratory values. A complete evaluation including von Willebrand factor multimers, ristocetin-induced platelet aggregation, factor VIII activity level, and a template bleeding time is necessary to correctly classify the patient so that optimal treatment may be given.

Transfusion medicine management for reconstructive spinal repair in a patient with von Willebrand's disease and a history of heavy surgical bleeding.

STUDY DESIGN: A case report of a multidisciplinary approach to a second reconstructive back surgery in a patient with von Willebrand's disease, flatback syndrome, and a history of heavy surgical bleeding is presented. OBJECTIVE: To review the perioperative planning and assessment of hemostasis and transfusion medicine management, including administration of Humate P, a Factor VIII preparation with high von Willebrand factor content. SUMMARY OF BACKGROUND DATA: Reconstructive spinal procedures may require significant transfusion support even in patients with normal preoperative hemostasis. In addition to the hemostatic problem caused by von Willebrand's disease, the reported patient requested minimal exposure to allogeneic blood products because of hepatitis C infection acquired from previous transfusions. METHODS: The multidisciplinary team included the patient, hematologist, blood bank medical director, anesthesiologist, and operating surgeon. Preoperative assessment showed a Type 2A von Willebrand's disease variant. A careful planning process included a test infusion of desmopressin and extensive autologous donations of red cells, plasma, and platelets, which were collected before the procedure. RESULTS: Anterior and posterior spine fusions were performed during a 14-hour procedure. Hemostasis and clinical response were excellent. Humate P was administered perioperatively as assessed by the baseline Factor VIII and von Willebrand's disease levels, the plasma volume, the half-life of infused Humate P, and the anticipated risk and tolerance for bleeding. The estimated blood loss was 5 L. Replacement included 9 units of autologous red cells, 6 units of autologous plasma, 2 autologous plateletpheresis collections, a single allogeneic plateletpheresis product, and 17,000 units of Humate P administered over the perioperative period. CONCLUSIONS: Using a careful multidisciplinary approach, excellent hemostasis can be achieved with minimal exposure to untreated allogeneic blood products during aggressive spinal surgery in a patient with a clinically significant congenital coagulopathy.