A critical evaluation of the available methods for the determination of factor VIII von Willebrand.

Von Willebrand factor (vWf) is the major component of the circulating factor VIII complex. The von Willebrand molecule includes factor VIII related antigen (VIIIR: Ag) which represents the molecular substrate of the von Willebrand activity expressed as Ristocetin cofactor (VIIIR:RCoF) activity. Several methods have been developed for VIIIR: Ag evaluation, among the first being the rocket-immunoelectrophoresis method of LAURELL. Radial immunodiffusion (MANCINI's method) was also used. Subsequently, radioimmunological assays, either as radioimmunoassay (RIA) or immunoradiometric assay (IRMA), were developed with improvements in sensitivity, so that levels of VIIIR: Ag lower than 0.1% of normal can be detected. More recently, an enzyme-linked immunosorbent assay (ELISA), characterized by the use of enzyme-conjugated antibody was proposed. This method shows a sensitivity similar to immunoradiometric methods but without using any dangerous reagent. Finally, a nephelometric method was proposed for factor VIII antigen evaluation. For a qualitative evaluation of von Willebrand factor crossed-immunoelectrophoresis and multimeric analysis can be used. In the first case, the use of precipiting antibodies against von Willebrand factor may demonstrate a peak with different characteristics related to the biochemical property of von Willebrand. Multimeric analysis in SDS-agarose gel electrophoresis followed by staining with labelled antifactor VIII antibodies gives information about different polymeric forms of circulating VIII/vW factor. Von Willebrand factor activity, expressed as its ability to induce platelet aggregation in the presence of the antibiotic Ristocetin, can be carried out using normal formalin fixed platelets, either with aggregometer or visual methods (glass slide test or tubes test and microtritation plate). The corrected evaluation of factor VIII complex by all these techniques together with the clotting activity assay allows a satisfactory study of factor VIII properties

Platelet aggregation and pseudothrombocytopenia induced by 1-desamino-8-D-arginine vasopressin (DDAVP) in type IIB von Willebrand's disease patient.

Our study shows that the thrombocytopenia described in type IIB von Willebrand's disease (vWd) after 1-desamino-8-D-arginine vasopressin (DDAVP) infusion is, at least partially, a pseudothrombocytopenia. There was a discrepancy in platelet counts in blood anticoagulated with EDTA (less than 10 x 10(3)/microliters) or citrate (55 x 10(3)/microliters) in one patient with type IIB vWd and chronic thrombocytopenia (80 x 10(3)/microliters) after DDAVP infusion. Furthermore, DDAVP induced a normalization of patient's prolonged bleeding time. Spontaneous platelet aggregation (SPA) observed in platelet-rich plasma before DDAVP infusion was inhibited completely by monoclonal antibodies which block binding of fibrinogen, vWf and fibronectin to GPIIb-IIIa. SPA was partially inhibited by a monoclonal antibody which blocks the binding of vWf to GPIb. After DDAVP, in contrast, SPA partially persisted in the presence of anti-GPIIb-IIIa monoclonal antibodies but was completely inhibited by anti-GPIb monoclonal antibody. Therefore GPIb and GPIIb-IIIa complex seem to play a different role in SPA before and after DDAVP infusion into type IIB vWd. I.F. 1,80

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

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

The use of antiproteolytic mixture fails to modify the abnormal von Willebrand factor pattern in myeloproliferative disease.

Abnormalities of von Willebrand factor, with reduction of higher molecular weight multimers, was described in patients with increased platelet number. We have studied twelve patients with myeloproliferative disease (9 patients with Essential Thrombocytosis and 3 patients with Polycythemia Vera). Blood samples were collected either with citrate and antiproteolytic mixture (EDTA 6 mM, Aprotinin 200 U/ml, N-ethylmaleimide 5 mM in sodium citrate 3.8%). VIIIR: RCoF was found decreased in all patients studied, using both anticoagulants, while VIIIR: Ag was within normal range. Higher molecular weight multimers were found decreased or absent both in samples collected with citrate and in those collected using the antiproteolytic mixture. These data suggested that von Willebrand factor abnormalities observed in myeloproliferative disease are not due to a proteolytic degradation that's supposed to happen in vitro during blood samples collection and manipulation

FACTOR-VIII VONWILLEBRAND-FACTOR ABNORMALITIES DURING L-ASPARAGINASE TREATMENT IN PATIENTS WITH ACUTE LYMPHOBLASTIC-LEUKEMIA

Factor VIII/von Willebrand factor (VIII/vWf) related properties were studied in 10 patients affected by acute lymphoblastic leukemia during L-asparaginase-vincristine-prednisone treatment. These properties remained within the normal range during the period of observation without any difference from the basal values. On the contrary, VIII:C activity was already increased before medication and showed gradual additional elevation during the observation period, reaching a peak 1 week after discontinuation of L-asparaginase administration. Crossed immunoelectrophoresis of vWf, performed weekly in 2 patients during the period of medication, demonstrated a normal pattern before the beginning of treatment, but an apparently faster migrating peak during L-asparaginase therapy, suggesting a qualitative abnormality of vWf. No abnormal bleeding tendency was found in any of the patients

Platelet fibrinogen: subcellular localization by means of immunofluorescent studies in normals and in congenital afibrinogenemia.

We have studied the site of fibrinogen localization in normal platelets and in the platelets of a patient with congenital afibrinogenemia (CA). The methods employed were: direct immunofluorescence technique (DIT) and indirect immunofluorescence technique (IIT). By means of the DIT normal platelets were shown to have a clear peripheral staining. Such staining disappeared after treatment with proteolytic enzymes and after specific blocking experiments. Such peripheral staining of platelets was absent in congenital afibrinogenemia even after fibrinogen infusion. By means of the IIT platelets were shown to have a considerable amount of fibrinogen. Such protein was demonstrated to represent an important part of platelet surface, since intact platelets were able to absorb completely a specific antifibrinogen antiserum