von Willebrand factor, von Willebrand factor-cleaving protease, and shear stress.

von Willebrand factor (VWF) is a multimeric plasma glycoprotein (GP) involved in platelet adhesion at the site of vascular damage, which acts as a bridge between the injured subendothelium and the platelet receptors. The multimeric structure of VWF allows it to support multiple interactions with platelets and endothelial components under high shear stress. Rapid flow conditions induce a conformational transition of the VWF molecule, thus allowing its functional binding domains to be exposed. A specific VWF-cleaving protease (ADAMTS-13) physiologically down regulates the multimeric size of newly released and circulating VWF in order to prevent unwanted platelet thrombus formation. The occurrence of microvascular platelet aggregation in thrombotic thrombocytopenic purpura, which is caused by an ADAMTS-13 deficiency, well-demonstrates the important role of the protease in regulating the adhesive activity of VWF. Better knowledge of VWF function would contribute to the development of novel anti-thrombotic strategies based on the selective inhibition of the VWF interaction with platelet receptors and endothelial components in areas of the circulation characterised by elevated fluid dynamic forces.

Identifying carriers of type 2N von Willebrand disease: procedures and significance.

The defective FVIII carrier function of von Willebrand factor (VWF) identifies type 2N von Willebrand disease (VWD), a variant with a pattern resembling hemophilia A. Type 2N characterization is based on the evaluation of the capacity of VWF to bind exogenous FVIII (VWF:FVIIIB). Here we report on a retrospective evaluation of hemostatic laboratory parameters most useful in detecting type 2N carriers. The diagnostic capacity of aPTT, FVIII, VWF:Ag, FVIII/VWF:Ag ratio, VWF:FVIIIB and VWF:FVIIIB/VWF:Ag ratio was evaluated in 21 type 2N VWD carriers. Twenty subjects were heterozygous for the R854Q mutation, one was heterozygous for the R760C missense mutation, which interferes with cleavage of the VWF propeptide. We found that prolongation of aPTT and decrease in FVIII and FVIII/VWF:Ag ratio were not frequent findings in type 2N carriers. The same was true for VWF:FVIIIB which was not always abnormal. On the contrary, VWF:FVIIIB/VWF:Ag ratio was always defective and its values were not related with FVIII and FVIII/VWF:Ag ratio or influenced by plasma VWF concentration. Given these results, we attribute the greatest significance to VWF:FVIIIB/VWF:Ag ratio in the diagnosis of type 2N defects, and only search for type 2N mutations, to validate the diagnosis, if the ratio proves abnormal.

Adhesion of MNC to extracellular matrix proteins following in vitro photochemotherapy.

BACKGROUND: Photochemotherapy is a safe and effective treatment for patients with drug-resistant severe GvHD. The technique involves the exposure of MNC to psoralen and UVA light (PUVA). We have investigated the effect of in vitro PUVA on MNC adhesion to extracellular matrix (ECM) proteins. METHODS: MNC were isolated from peripheral blood (PB) and umbilical cord blood (UCB), and treated with PUVA. After labeling by a chemiluminescent probe, MNC were plated on ECM proteins (collagen, fibronectin, vitronectin and laminin) and the number of adherent cells was measured. RESULTS: Untreated MNC from both PB and UCB showed a similar adhesion to the substrates. As a consequence of exposure to PUVA, most of PB samples showed significantly enhanced adhesion to the ECM proteins; on the other hand, UCB-recovered MNC did not significantly modify their adhesion. DISCUSSION: MNC adhesion to ECM components is mediated by integrins, a family of cell membrane receptors; the ligand-binding affinity of certain integrins may be modulated by different stimuli. PUVA treatment of PB-recovered MNC may induce the up-regulation of the ligand-binding affinity of the integrins involved in the adhesion to ECM proteins. The finding of unmodified UCB cell adhesion after PUVA, may be related to the functional immaturity of lymphocytes at birth.

[Recovery of transplantable hematopoietic progenitor cells from the umbilical cord blood]. / Recupero di progenitori emopoietici trapiantabili da sangue di cordone ombelicale.

Cord blood (CB) is a source of transplantable hematopoietic progenitor cells (HPC); it represents an alternative to bone marrow to restore hematopoiesis in patients affected by malignant and non-malignant disease. Therefore, large-scale CB banks would be a natural complement to bone marrow donor registries. Storage of unmanipulated whole CB units requires a great number of liquid nitrogen containers. Separation of leukocytes allows CB storage in smaller space, thus lowering banking costs; unfortunately, CB processing may cause significant losses of stem/progenitor cells. We describe here a procedure for erythrocyte removal from CB units by 1 xg sedimentation on Emagel, a gelatin-based colloidal compound commonly used as plasma expander. The erythrocyte-depleted supernatant was collected and then centrifuged to recover the leukocyte pool. We evaluated erythrocyte depletion and leukocyte recovery after different sedimentation time (30, 45 and 60 min), on 139 CB units collected at delivery. All the considered parameters were improved by increasing sedimentation time. Erythrocyte depletion at 60 min was 86.0% and we recovered 93.3% of CD34+ cells. The proposed CB-processing method allowed us to collect a satisfactory amount of HPC in view of stem cell transplantation; it may have a potential role in UCB banking.

Quantitative and qualitative evaluation of blood salvaged after extracorporeal circulation (ECC) in paediatric heart surgery. Study of biochemical, morphological and structural variations of RBC after ECC and after salvaging of ECC circuit priming blood.

The salvaging of ECC circuit priming blood is essential for reducing the morbidity related to homologous blood transfusions and the importance of this technique is inversely proportionate to the age and weight of the child. In infants, the washing and centrifugation of blood not only drastically reduce the risk of contracting blood-transmitted diseases and cut management costs, but are also of considerable hemodynamic importance, producing a rapid normalization of the patient's hematocrit and hemoglobin and balancing the O2 consumption/demand ratio. The marketing of miniaturized salvagin devices with 55 ml bowls by Dideco has made possible the recovery of small quantities of blood, so as to normalise the hematic crisis and permit the application of total hemodilution in low-weight patients. The salvaged blood shows an average hematocrit of 52.7+/-9.7% (max 68.1%) and an average hemoglobin of 17.6 +/- 2.9 g/dl (max 20.7 g/dl), and maintains its structural components, osmotic resistance, concentration of intraerythrocytic hemoglobin and mean corpuscular hemoglobin all intact. Washing with isoosmotic and isoionic hydroelectrolytic solutions normalizes the ionic situation in the post-operative period and activated blood salvaging after Extracorporeal Circulation. The use of solutions without nutritional substances results however in a considerable fall in the number of enzymes in the intraerythrocytic metabolic glucide chain (G6PDH: -40.7 +/- 14.3% p<0.001), (PK: -23.8 +/- 20.5% p<0.03). This drop may be responsible for erythrocytic morphological alterations (echinocytic change) and probably for the release of hemoglobin from the red blood cells. Washing with isoionic, isoosmotic solutions containing G5% and adenine could, at least in theory, improve the quality of the salvaged blood, by normalizing the morphology and the volume of the RBC and by increasing the hematocrit.

Processing of human cord blood by three different procedures for red blood cell depletion and mononuclear cell recovery.

BACKGROUND AND OBJECTIVES: Human cord blood (CB) is an important source of stem cells which may be used for hematopoietic reconstitution as an alternative to bone marrow transplantation. Banking of CB would be accomplished by removing red blood cells (RBC) and plasma from CB collections. Our aim was to compare three different procedures for CB processing. MATERIALS AND METHODS: Poligeline, hydroxyethyl starch gel (HES) and gelatin were used as separation media in processing 79 CB units for RBC depletion and mononuclear cell (MNC) recovery. RESULTS: The best MNC recoveries were obtained performing the HES- and the gelatin-based procedures (80.9 and 84.7%, respectively), but the gelatin procedure allowed us to obtain the highest RBC depletion (96.4%); CD34+ cell recovery was higher using HES or gelatin as separation media (85.6 and 85.9%, respectively). CONCLUSION: The best results, as far as RBC removal and MNC recovery are concerned, were obtained by using gelatin as RBC sedimentation medium. Gelatin is a low-cost, animal-derived reagent, which has been successfully used for CB transplantation; the procedure is simple to perform and appears to be suitable for large-scale banking in view of CB transplantation.

Leukocyte recovery from umbilical cord blood by poligeline.

Umbilical cord blood (UCB) collected at delivery is a source of transplantable stem/progenitor cells; it represents an alternative to bone marrow to restore hematopoiesis in patients affected by malignant and non-malignant disease. Therefore, large-scale UCB banks would be a natural complement to bone marrow donor registries. Storage of unmanipulated whole UCB units requires a great number of liquid nitrogen containers. Separation of leukocytes allows UCB storage in smaller space, thus lowering banking costs; unfortunately, UCB processing may cause significant losses of stem cells. We report about the use of poligeline to remove erythrocytes from UCB units. After erythrocyte sedimentation at 1xg (30' or 40') or 50xg, leukocyte-rich supernatant was collected and centrifuged to recover the leukocyte pool in view of stem cell transplantation. Erythrocyte depletion was always satisfactory, ranging from 82.6% to 88.9%, but 1xg sedimentation for 40' enabled us to achieve the best CD34+ cell recovery (mean value 80.5%). The proposed UCB-processing method allowed us to lower the final sample volume down to 1/10 of the initial one, in this way making UCB banking feasible. Erythrocyte depletion took place directly in the collection bag, thus reducing microbial contamination risk.

Two-dimensional analysis of the structure of human von Willebrand factor.

Human von Willebrand factor (vWF) is synthesized as an extra large polymer; then, it is converted to lower molecular weight plasma multimers, originally composed of intact 225-kDa subunits, by a metalloproteinase. Proteolysis generates two fragments of 140-and 176-kDa, which originate from cleavage of peptide bond Tyr842-Met843 and which represent vWF residues 1-842 and 843-2050, respectively; a very small amount of 189-kDa fragment can also be found in normal plasma. We describe here a two-dimensional (2-D) method to analyze plasma vWF structure.

Detection of megakaryocyte colonies in plasma clot cultures by immunoenzymatic staining.

In vitro induced megakaryocytic differentiation/maturation of megakayocyte (meg) progenitors represents an important tool for investigating cytokine-induced in vitro thrombocytopoiesis. We have developed an assay which allows the in situ study of human meg progenitor-derived colonies, cultured on a plasma clot in the presence of cytokines. Plates were immunostained by using an anti-alpha IIb beta 3 monoclonal antibody and an alkaline phosphatase-labeled secondary antibody. alpha IIb beta 3-bearing cells were stained an intense red and were clearly differentiated from the negative cells. Processed plates were stable for some weeks at 4 degrees C. The described procedure is easy to perform and allowed us to enumerate the meg colonies and assess colony morphology and cell ploidy.

Interaction of the von Willebrand factor with platelets and thrombosis.

The human von Willebrand factor (vWf) is a multimeric glycoprotein present in plasma, platelets, endothelial cells and subendothelium and synthesized in endothelial cells and megakaryocytes. vWf plays a pivotal role in the mechanisms of blood clotting and platelet thrombus formation; quantitative and qualitative abnormalities of vWf cause the most common congenital bleeding disorder in man, the von Willebrand disease. vWf stabilizes factor VIII and interacts with subendothelial components and with platelet membrane receptors. The multimeric structure of vWf provides an array of binding sites which allows multivalent interactions with its ligands, thus supporting the formation of stable platelet aggregates at the site of vascular injury, particularly under flow conditions characterized by high shear stress. In the last years, remarkable progress has been made toward understanding the structure of vWf protein and gene, and the elucidation of many structure-function relationships, which may result in improved therapeutic intervention for vWD patients, and in the development of effective strategies for antithrombotic therapy.

von Willebrand factor: biological function and molecular defects.

The human von Willebrand factor (vWF) plays a pivotal role in the mechanisms of blood clotting and platelet thrombus formation; it also binds and stabilizes factor VIII procoagulant protein. The biological functions of vWF are dependent on distinct molecular domains responsible for the specificity and affinity for ligands. The multimeric structure of vWF provides an array of binding sites that allow multivalent interactions, thus supporting the formation of stable platelet aggregates at the site of vascular injury, particularly under flow conditions characterized by high shear stress. Quantitative and qualitative abnormalities of vWF cause the most common congenital bleeding disorder in humans, the von Willebrand disease (vWD). This review will provide an update on the recent advances toward the elucidation of structure-function relationships and the detection of molecular defects leading to vWD and will highlight the revised classification of vWD.

Platelet antibody detection in pediatric immune thrombocytopenic purpura: evaluation of three screening methods.

BACKGROUND AND OBJECTIVES: Immune thrombocytopenic purpura (ITP) is a common hematologic disorder, two forms of which occur in children. The detection of circulating platelet antibodies is helpful in diagnosis. MATERIALS AND METHODS: We evaluated three different immunological methods for detecting platelet antibodies in the serum of children with ITP. These were: a solid-phase red-cell adherence test (SPRCA), an enzyme immunoassay (EIA), and an immunofluorescence test (PSIF). RESULTS: The sensitivity of the methods in detecting IgG antibodies ranged from 28.1 (EIA) to 39.4% (SPRCA). We also looked for IgM antibodies by PSIF, thus raising the sensitivity of this test from 32.0 to 40.0%. A combination of two tests (SPRCA and EIA) allowed us to detect 61.8% positive samples. By doing all three tests, we obtained 71.3% positive samples. Finally, we reached 73.5% by adding PSIF for IgM. We found a higher frequency of circulating antibodies in both acute and chronic ITP at onset than in clinical remission. There were a few positive sera in chronic ITP, but not in the acute form in remission. CONCLUSION: The individual tests each have a relatively low sensitivity, but the combination of all three increases the diagnostic effectiveness. The finding of platelet antibodies during remission may predict evolution toward a systemic autoimmune state.

[Disintegrins: potent inhibitors of platelet aggregation]. / Le disintegrine: potenti inibitori dell'aggregazione piastrinica.

Disintegrins are a family of highly homologous polypeptides purified from snake venoms, which contain the arginine-glycine-aspartic acid (RGD) sequence. The RGD tripeptide acts as an integrin recognition sequence; it is also present on several proteins involved in cell adhesion such as fibrinogen, fibronectin, von Willebrand factor, and collagen. Disintegrins can therefore competitively inhibit integrin-ligand interactions: they block fibrinogen binding to its platelet receptor, alpha IIb beta 3: hence they are potent platelet aggregation inhibitors. Disintegrins are up to 2000 times more potent than short synthetic linear RGD-containing peptides in blocking fibrinogen-dependent platelet aggregation. Likely, the amino acids surrounding the RGD sequence and intrachain disulphide bridges force the RGD sequence in an appropriate conformation which accounts for the high, but variable, platelet inhibitory activity exhibited by disintegrin molecules. Disintegrins block the adhesive functions of the RGD-dependent integrins present on different cell types in different tissues: for this reason they are not alpha IIb beta 3-specific. A single disintegrin polypeptide, barbourin, was found to be a specific alpha IIb beta 3 antagonist: unlike all other disintegrins it contains the lysine-glycine-aspartic acid (KGD) sequence instead of the RGD one, which solely imparts alpha IIb beta 3 specificity to the molecule. This finding led to the synthesis of small, conformationally constrained KGD-containing peptides, which proven to be specific and potent inhibitors of alpha IIb beta 3 function; these compounds are presently undergoing evaluation in clinical trials as antithrombotic agents.