Analysis of von Willebrand factor in platelets of patients with various forms of von Willebrand disease: Is there a clinical relevance?

Besides the investigation of coagulation factor VIII:c and von Willebrand factor in plasma, vWF antigen and vWF collagen-binding activity in platelets of 24 patients with various forms of von Willebrand disease were analysed. No platelet vWF:Ag or vWF:CBA was detectable in type 3 patients (n = 4). In contrast 6 out of 7 patients with type 2 vWD had normal or increased vWF levels. Two type 1 patients (out of n = 13) with low von Willebrand factor in platelets had no increased bleeding tendency. In two other individuals with normal amounts of von Willebrand factor in platelets and low plasmatic vWF and factor VIII:c, more frequent bleeding episodes reflecting the low plasmatic levels were observed in a long-term follow-up. Conclusion In our patients, bleeding history corresponded to plasmatic levels of FVIII:c and vWF.     

Management of severe chronic thrombocytopenia in von Willebrand's disease type 2B

Two patients with a long history of unexplained thrombocytopenia, eventually diagnosed with von Willebrand''s disease (vWD) type 2B are reported. In one patient with platelet counts of 80× 10⁹/l 1-desamino-8-D-arginine vasopressin (DDAVP) had a favourable effect during bleeding episodes. The second patient received intermediate purity von Willebrand''s factor (vWF)/factor VIII concentrate (Haemate HS), which helped haemostasis during tooth extraction. It increased platelet counts from 15 to 30 × 10⁹/l, whereas platelet transfusions produced no increase, nor prevented severe bleeding during abdominal surgery. Thus the treatment of vWD type 2B might depend on the degree of thrombocytopenia. It is recommended that in patients with mild to moderately decreased platelet counts, DDAVP treatment can be tried, whereas in patients with severely decreased platelet counts intermediate purity vWF/factor VIII concentrate substitution is preferred.
In addition, vWD type 2B should be considered in the differential diagnosis of any child with chronic thrombocytopenia as the treatment strategy is different.

     

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.     

Management of severe chronic thrombocytopenia in von Willebrand's disease type 2B

Two patients with a long history of unexplained thrombocytopenia, eventually diagnosed with von Willebrand's disease (vWD) type 2B are reported. In one patient with platelet counts of 80 x 10(9)/l 1-desamino-8-D-arginine vasopressin (DDAVP) had a favourable effect during bleeding episodes. The second patient received intermediate purity von Willebrand's factor (vWF)/factor VIII concentrate (Haemate HS), which helped haemostasis during tooth extraction. It increased platelet counts from 15 to 30 x 10(9)/l, whereas platelet transfusions produced no increase, nor prevented severe bleeding during abdominal surgery. Thus the treatment of vWD type 2B might depend on the degree of thrombocytopenia. It is recommended that in patients with mild to moderately decreased platelet counts, DDAVP treatment can be tried, whereas in patients with severely decreased platelet counts intermediate purity vWF/factor VIII concentrate substitution is preferred. In addition, vWD type 2B should be considered in the differential diagnosis of any child with chronic thrombocytopenia as the treatment strategy is different.     

Relative value of diagnostic studies for von Willebrand disease.

Laboratory tests recommended to screen patients with mucosal bleeding for hemostatic disorders generally include determinations of prothrombin time, partial thromboplastin time, platelet count, and bleeding time. To determine the best tests to identify patients with von Willebrand disease (vWD), we reviewed the laboratory studies of 24 children with vWD and performed receiver operating characteristic analysis on the diagnostic studies. The vWD disease diagnostic tests included determinations of vWF activity (ristocetin cofactor activity), vWF factor antigen, and factor VIII procoagulant (VIII:c). The diagnosis of vWD required the presence of a personal and family history of bleeding symptoms and a documented abnormality of vWF activity or vWF antigen. vWF activity, vWF antigen, factor VIII:c and blood type were determined in 104 symptom-free children. There were no differences between patients and normal subjects for age, gender, or blood type. The bleeding time was abnormal in 43%, the partial thromboplastin time was abnormal in 25%, and either one or both were abnormal in 58% of the patients. The vWF activity, vWF antigen, and factor VIII:c were abnormal in 79%, 58%, and 33%, respectively. Receiver-operated-characteristic analysis showed the vWF activity to be superior to either the vWF antigen or factor VIII:c in establishing the diagnosis of vWD. The combination of the activity, bleeding time, and partial thromboplastin time successfully identified 92% of the patients as abnormal. Determination of vWF activity should be included routinely in the evaluation of hemostasis in children with symptomatic disease.     

Upshaw-Schulman Syndrome in Two Siblings

Upshaw-Schulman syndrome is a rare disease, and familial occurrence has not been reported. In this paper, two Japanese brothers, aged 2 and 6 years, with microangiopathic hemolytic anemia, thrombocytopenia and fragmented red cells from the newborn period are reported. Unusually large von Willebrand factor (vWF) multimers were found in the plasma samples from both cases during remission, while the quantities of the unusually large vWF multimers decreased greatly at a low platelet count. They were temporarily relieved from hemolytic anemia and thrombocytopenia by infusions of plasma from normal donors. It appears that our patients lack an unidentified plasma factor, which is genetically determined.     

Acquired von Willebrand's syndrome with lupus-like serology

We describe a 12-year-old boy with acquired von Willebrand's syndrome, who also had various autoantibodies. He presented with recent hemorrhagic symptoms and a prolonged bleeding time. Hemostatic studies revealed severely reduced levels of factor VIII procoagulant activity (VIII:C), von Willebrand's factor (vWF) antigen (vWF:Ag), and ristocetin cofactor activity (RCoF). An inhibitor that could be detected in the patient's plasma moderately decreased the levels of vWF:Ag in normal plasma, but did not interfere with the measurement of VIII:C or RCoF. Following the infusion of cryoprecipitate, half-lives of VIII:C, vWF:Ag, and RCoF were markedly reduced. 1-Deamino-8-D-arginine vasopressin infusion induced normalization of the prolonged bleeding time and caused a marked increase in VIII:C, vWF:Ag, and RCoF. Prior to treatment, there was a uniform reduction of all the multimers of plasma vWF in sodium dodecyl sulfate agarose gel electrophoresis. Following prednisone therapy, clinical and hemostatic findings were improved, and the multimeric patterns of vWF were normalized. These findings suggest that the low levels of all three parameters of factor VIII and all the multimers of plasma vWF in the patient are caused by rapid elimination of factor VIII complex from the circulation.     

Advances in the genetics and treatment of von Willebrand disease

von Willebrand disease (VWD) is a bleeding disorder caused by quantitative (type 1 and 3) or qualitative (type 2) defects of von Willebrand factor (VWF). The mechanisms of most inherited VWD types have been recently elucidated by genetic and molecular diagnosis, but the phenotypic tests based on measurements of plasma and platelet VWF, the ability of VWF to interact with its platelet receptor, and the analysis of the multimeric composition of VWF are always essential to identify patients with different VWD subtypes. The aim of treatment is to correct the dual defects of hemostasis, ie, abnormal coagulation expressed by low levels of factor VIII (FVIII) and abnormal platelet adhesion expressed by prolonged bleeding time (BT). Desmopressin is the treatment of choice in most patients with type 1 and type 2 VWD, who account for 60 to 70% of cases. In type 3 and in some severe forms of type 1 and type 2 VWD, desmopressin is not effective, and it is necessary to resort to plasma concentrates containing FVIII and VWF. Treated with virucidal methods, these concentrates are effective and currently safe, but they do not always correct the BT defect. Platelet concentrates or desmopressin can be used as adjunctive treatments when poor correction of the BT after concentrates is associated with continued bleeding.     

Regulated release of VWF and FVIII and the biologic implications

von Willebrand factor (VWF) performs a critical function in platelet binding at the site of vascular injury and also serves as the carrier protein for coagulation factor FVIII (FVIII), protecting it from proteolytic degradation in plasma. Both proteins undergo rapid, regulated release in response to DDAVP administration in patients with mild hemophilia A or von Wille-brand disease. Here, we attempt to summarize our current understanding of the establishment of the regulated storage pool of VWF and FVIII. The data presented indicate that regulated secretion of both proteins occurs only if there is endogenous synthesis of FVIII together with VWF.     

Management of type 2b von Willebrand disease in the neonatal period

Von Willebrand disease (VWD) is the most common inherited bleeding disorder, affecting one in 1,000 people. Type 2b VWD is a less common subtype caused by a gain‐of‐function mutation in von Willebrand factor (VWF) that leads to the formation of large, ineffective VWF‐platelet multimers in circulation. This unique pathophysiology creates diagnostic and treatment dilemmas. There is limited information on the management of type 2b VWD in the neonatal period. This report describes the management of a neonate with type 2b VWD with an emphasis on the added benefit of concomitant platelet transfusion and factor replacement therapy over factor replacement therapy alone.     

Whole blood platelet deposition on extracellular matrix under flow conditions in preterm neonatal sepsis

Platelet function in preterm infants with sepsis was evaluated by measuring their adhesion and aggregation properties using the Cone and Plate(let) Analyser. This may lead to earlier detection of bleeding tendency in septic infants. Platelet function was investigated in 54 preterm infants, of whom 32 had proven neonatal sepsis and 22 were healthy matched controls. Citrated whole blood was subjected to shear stress (1300 s(-1)) for 2 min on tissue culture plates precoated with subendothelial extracellular matrix (ECM). The percentage of ECM surface covered with platelets and the average size of the ECM-bound platelet particles were determined with an image analyser. Assays for von Willebrand factor (vWF) antigen, ristocetin co-factor, and vWF collagen binding activity (CBA) were performed on samples from an additional 47 preterm infants: 38 healthy and 9 septic. Platelets of the preterm infants with sepsis displayed lower adhesion than those of the healthy controls. Mean surface coverage was 16.9+/-8.2% for the septic infants, 15.4+/-7.9% for the septic infants after exclusion of those with coagulase-negative staphylococci sepsis, and 20.8+/-9.6% for the healthy group ( P<0.05). Platelet aggregation, vWF antigen, ristocetin co-factor, and CBA levels were similar between the septic and healthy groups. The most significant factor influencing surface coverage was the presence of sepsis. CONCLUSION: platelet adhesion to extracellular matrix is significantly lower in septic preterm infants than in healthy preterm infants. Intrinsic platelet properties, rather than the concentration or activity of plasma von Willebrand factor, may be responsible for this difference. Surface coverage obtained by the collagen binding activity test under flow conditions, which represents platelet adhesion, may be an earlier, more sensitive indicator of bleeding tendency in neonatal sepsis than decreased platelet count.     

A case of inherited type 1 and type 2A von Willebrand disease confirmed by diagnostic exome sequencing

A 10‐year‐old male and his family members visited a pediatric hematology clinic due to coagulopathy. Laboratory tests indicated von Willebrand disease (vWD) in all the family members. We conducted diagnostic exome sequencing for confirmation. The patient was confirmed to be a compound heterozygote for vWD: c.2574C > G (p.Cys858Trp) from his father (known variant of vWD type 1) and c.3390C > T (p.Pro1127_Gly1180delinsArg) from his mother (variant known to result in exon 26 skipping in vWD type 2A). He was managed with factor VIII and von Willebrand factor complex concentrate during palatoplasty due to bleeding despite pre‐operative desmopressin injection. The operation was completed successfully.     

Shorter PFA-100® closure times in neonates than in adults: role of red cells, white cells, platelets and von Willebrand factor

In neonates, despite poor platelet function in various in vitro tests, closure times (CTs) in PFA-100^® measurements are shorter than in adults. Neonates have a higher polymeric von Willebrand factor (vWF). They also have a higher haematocrit and higher white blood cell count than adults, which may interfere with the evaluation of platelet and vWF function by means of the PFA-100 in neonates. To assess the role of different blood constituents on neonatal CTs, red blood cell, platelet and white blood cell counts in cord blood were modified. These modifications did not provide any evidence that the difference in number between adult and neonatal blood cells was responsible for shorter neonatal CTs. In further experiments, platelets and/or vWF were inhibited by means of abciximab and anti-vWF antibody, and mixing experiments with neonatal platelet-rich and platelet-poor plasma were performed. The results showed that short cord blood PFA-100 CTs were caused by a constituent of neonatal platelet-poor plasma, probably the neonatal high multimeric vWF. Conclusion: This study demonstrates that CTs in neonates are dependent on the same components, platelets and vWF, as in adults, making it likely that the PFA-100 can be used in neonates in the same way as in adults to investigate platelet and vWF function.     

von Willebrand factor-collagen binding activity is increased in newborns and infants

ABSTRACT. von Willebrand factor (vWF) antigen (vWF:Ag) and vWF-collagen binding activity (vWF:CBA) were measured in plasma by parallel quantitative ELISAs in normal newborns and infants ( n =71). The medians for vWF:Ag (IUjml) and vWF:CBA (Ujml), respectively, were 1.46 and 1.91 for 2-7 day-old (n = 43), 1.22 and 1.40 for 2-4 week-old (n = 14), 1.22 and 1.15 for 2-6-month-old (n = 14) infants and 0.98 and 1.08 (n = 36) in normal adults. Elevated levels of vWF:Ag, but particularly vWF:CBA were seen for up to 4 weeks of life reaching adult levels between 2 and 6 months of life. The elevated levels of the vWF parameters indicate that caution should be exercised when interpreting laboratory data and diagnosing von Willebrand disease in newborns and young infants and warrant the use of age-specific reference ranges. The efficient haemostasis observed during early neonatal life may in part be due to the increased ability of vWF to interact with collagen.     

Decreased clearance of von Willebrand factor in a patient with type 2B von Willebrand disease following development of immune thrombocytopenia

We report a case of concurrent type 2B von Willebrand disease (VWD) and immune thrombocytopenia (ITP). The patient had characteristic loss of von Willebrand factor (VWF) high molecular weight multimers (HMWM) but a normal platelet count in the initial 8 years after diagnosis of type 2B VWD. When he developed severe thrombocytopenia, however, both his VWD indices and VWF HMWM normalized. As his platelet count increased, he again lost the HMWM and his VWD indices decreased. These results suggest that the severe thrombocytopenia led to decreased clearance of VWF, especially the HMWM.     

Increased neonatal platelet deposition on subendothelium under flow conditions: the role of plasma von Willebrand factor

In vitro platelet function of umbilical cord blood and neonatal peripheral vein blood from full-term newborns was compared with that of adults. Citrated whole blood was subjected to shear stress (1300 s(-1)) on subendothelial extracellular matrix (ECM)-coated wells in a cone and plate(let) analyzer. Adhered platelets on the ECM were quantitated by image analyzer. Both umbilical cord and neonatal peripheral blood platelets demonstrated more extensive adhesion than adult platelets, and similar aggregate formation on ECM. The ability of neonatal platelets to form aggregates on ECM was confirmed by scanning electron microscopy. Similar activation of neonatal and adult platelets after subjection to shear stress, in the suspension phase, was established by flow cytometry, which showed an increase in fibrinogen binding and a decrease in glycoprotein Ib expression on platelet membrane. The difference in adhesion rates between neonatal and adult platelets was preserved even when the hematocrit level of the neonatal blood was adjusted to that of adults. Reconstitution of neonatal or adult platelet-rich plasma with autologous or heterologous red packed cells yielded no change in adhesion and aggregation. When von Willebrand factor-covered plates were used to prevent deposition of plasma von Willebrand factor on the surface, no difference in platelet adhesion was seen between neonatal and adult blood. In conventional aggregometry assay, the response to ristocetin of washed platelets of either neonatal or adult source was higher on addition of plasma from neonates than from adults. Our data suggest that the extensive neonatal platelet deposition on ECM is mediated by plasma von Willebrand factor, which is known to be more multimerized and, therefore, more active in neonates than in adults. This mechanism may provide balanced primary hemostasis in neonates despite the platelet hyporeactivity to agonists without application of shear stress.     

Influenza‐associated thrombotic microangiopathy with unbalanced von Willebrand factor and a disintegrin and metalloproteinase with a thrombospondin type 1 motif,member 13 levels in a heterozygous protein S‐deficient boy

Influenza infections often cause pneumonia, but there is limited information on thrombotic microangiopathy (TMA) in these circumstances. We report the case of an 11‐year‐old boy who developed TMA during the acute phase of H1N1 influenza. Plasma von Willebrand factor (VWF) was elevated, whereas a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) activity was mildly reduced in the absence of ADAMTS13‐neutralizing autoantibody, resulting in low ratio of ADAMTS13 to VWF. The patient was treated intensively, including plasma exchange, and he recovered from the TMA. He developed pulmonary embolism (PE), however, after removal of the central venous catheter. The findings suggested that influenza‐associated cytokines enhanced the release of unusually large VWF multimers from vascular endothelial cells and promoted the formation of platelet thrombi and TMA. Subsequent analysis further indicated the presence of familial protein S deficiency, and it seemed likely that the PE was more related to this heterozygous protein S defect.     

新生儿硬肿症血浆血管性假血友病因子的意义

目的 探讨新生儿硬肿症血浆血管性假血友病因子(vWF)的意义.方法 采用免疫浊度法检测39例新生儿硬肿症和11例正常新生儿血浆vWF水平.结果 硬肿症组血浆vWF明显高于对照组(P＜0.005),轻度硬肿组与对照组比较无显著性差异(P＞0.05),中、重度硬肿组血浆vWF均明显高于对照组(P均＜0.05),且血浆vWF升高与硬肿症程度相一致;血浆vWF与血pH呈负相关(r=-0.808 P＜0.001).结论 中、重度硬肿症体内存在以高凝为主的早期DIC,血管内皮细胞损伤参与其病理生理过程.     

Platelet function in autoimmune (idiopathic) thrombocytopenic purpura

Platelets play an essential role in the formation of haemostatic plugs. The quantitative defect of platelets in autoimmune (idiopathic) thrombocytopenic purpura (ITP) can result in bleeding complications, but most ITP patients have platelets with normal or enhanced function. Platelets in ITP are large, young, so-called "stress" platelets with increased platelet-associated autoimmune antibody (immunoglobulin G). Young stress platelets are more functional platelets, and their presence may account for bleeding times in ITP patients that are shorter than would be predicted on the basis of the patients' (low) platelet counts. Some ITP patients have significant mucocutaneous bleeding with platelet counts >50 × 10⁹ l⁻¹; this may be due to qualitative platelet dysfunction (e.g. brought about by inhibitory antiplatelet autoantibodies).     

Formation of hydroxyeicosatetraenoic acids (HETE) in blood from adults versus neonates: reduced production of 12-HETE in cord blood

Hydroxyeicosatetraenoic acids (HETE) are major arachidonic acid metabolites of a number of cells found in blood and blood vessels. These products have been implicated in physiologic responses as diverse as platelet aggregation, cell migration, and cell proliferation. Using a sensitive and specific assay, GC/selected ion monitoring after high-performance liquid chromatography separation, we have measured the levels of three HETE isomers of biologic significance 12-HETE, 15-HETE, and 5-HETE in plasma, serum and stimulated serum (formed in the presence of arachidonic acid and calcium ionophore), obtained from normal adults and cord blood from normal neonates. Whereas there were no significant differences between the two groups for 5- or 15-HETE in any of the samples, stimulated serum from adults produced 12 times as much 12-HETE when compared to cord blood. When platelets were isolated from adult and cord blood, 12-HETE production by neonatal platelets, stimulated with 10 microM arachidonic acid, was less than one-fourth that of adults. Although no role for 12-HETE in normal platelet responses has yet been established, it has been reported that those individuals with myeloproliferative syndromes who demonstrate a concomitant decrease in platelet 12-HETE synthetic ability have an increased bleeding tendency. It needs to be further evaluated if this already depressed level of 12-lipoxygenase in neonatal platelets may contribute to pathologic bleeding in those infants subjected to additional stress (such as prematurity or birth asphyxia).