Spontaneous platelet aggregation in type IIB Tampa von Willebrand disease is inhibited by the 52/48-kDa fragment of normal von Willebrand factor, which contains the GPIb binding domain

The association of Type IIB von Willebrand disease (vWD) with chronic persistent thrombocytopenia and spontaneous platelet aggregation has recently been recognized. It has been shown that IIB von Willebrand factor (vWF) can initiate platelet aggregation by binding to the platelet glycoprotein (GP) lb receptor and inducing exposure of the GpIIb/IIIa fibrinogen receptor. In this study we demonstrate the increased binding of Type IIB Tampa vWF with normal platelets when compared with nonthrombocytopenic Type IIB vWF. Studies further demonstrate that spontaneous platelet aggregation initiated by IIB Tampa vWF can be blocked by a 52/48-kDa fragment of normal vWF, which contains the binding domain.     

Von Willebrand disease associated with familial thrombocytopenia and increased ristocetin-induced platelet aggregation

Two cases of von Willebrand disease (vWD) associated with familial thrombocytopenia were reported. The proband (daughter) and her father showed thrombocytopenia with large platelets and decreased von Willebrand factor activity (VIIIR:WF). Factor VIII procoagulant activity (VIII:C) and factor VIII-related antigen (VIIIR:AG) were normal, but both patients revealed an increased ristocetin-induced platelet aggregation and a qualitative abnormality of the factor VIII protein, which was characterized by fast electrophoretic mobility of VIIIR:AG and an abnormal elution of factor VIII-related activities on Sepharose 2B. DDAVP was hemostatically effective even in this thrombocytopenic patient undergoing a dental extraction.     

Type 2B Hiroshima: a variant of von Willebrand disease characterized by chronic thrombocytopenia and the presence of all von Willebrand factor multimers in plasma.

Type 2B von Willebrand disease (vWD) is a von Willebrand factor (vWF) subtype with increased binding affinity for platelet glycoprotein (GP) Ib and is characterized by increased ristocetin-induced platelet agglutination at low concentrations of ristocetin. Usually there are no high molecular weight multimers of vWF, and platelet counts are within normal ranges in patients with type 2B vWD. We identified a variant of type 2B vWD showing the full range of vWF multimers in plasma accompanied by thrombocytopenia, which seemed to be caused by circulating platelet aggregation. Since the A1 domain and surrounding region of vWF alleles, in which mutation sites are known to be clustered in type 2B vWD, appeared normal on nucleotide sequencing, this increased binding affinity of vWF for GPIb may be due to a novel mechanism differing from that which usually underlies type 2B vWD.     

Discrepancy between IIA phenotype and IIB genotype in a patient with a variant of von Willebrand disease

Type IIA and IIB von Willebrand disease (vWD) result from qualitative abnormalities of von Willebrand factor (vWF) characterized by an absence in plasma of high molecular weight vWF multimers and an abnormal reactivity of vWF towards platelet glycoprotein (GP) Ib, which is decreased in type IIA and increased in type IIB. In this report, we describe the case of a patient having a IIA vWD phenotype associated with an intermittent thrombocytopenia atypical in this subtype but observed in type IIB vWD. The patient plasma vWF showed an absence of high molecular weight and intermediate multimers and had a decreased binding capacity to GPIb. The affinity of botrocetin was normal for plasma vWF from the propositus. Analysis of the propositus vWF gene showed the presence of a substitution Val 551 to Phe of the mature vWF subunit. This mutation is localized within a 509-695 disulphide loop of the vWF that plays an important role in the binding to GPIb and is where most of the molecular defects described so far were associated with type-IIB vWD. We have reproduced the Val 551 Phe substitution onto the vWF cDNA, expressed it in COS-7 cells, and performed structural and functional analysis of the mutant recombinant protein (rvWFPhe 551). The rvWFPhe 551 had a normal multimeric structure and showed the capacity to spontaneously interact with GPIb. Botrocetin had a decreased affinity for rvWFPhe 551. In conclusion, the Val 551 Phe mutation modifies the affinity of vWF for platelet GPIb, as does a type IIB mutation, and may be responsible for the thrombocytopenia of the patient and the clearance of the high molecular weight and intermediate- sized multimers of vWF from the plasma. The study of the rvWFPhe 551 has confirmed the discrepancy between the IIA phenotype and the IIB genotype of the patient.     

Botrocetin- and polybrene-induced platelet aggregation in platelet-type von Willebrand disease

It has been reported that botrocetin, a Bothrops venom factor, induces platelet aggregation dependent on von Willebrand factor (vWF), and that platelet aggregation induced by Polybrene, a synthetic polycation, is enhanced by vWF. This report describes the platelet aggregability on stimulation with botrocetin and Polybrene in four patients with platelet-type von Willebrand disease (vWD) who showed increased platelet aggregation with low concentrations of ristocetin as the result of a platelet abnormality. Enhanced platelet aggregability with botrocetin was observed in platelet-rich plasma (PRP) from the patients. Platelet aggregation induced by botrocetin in a mixture of normal washed platelets and patient plasma was either decreased or normal, being dependent on the amount of plasma vWF. In contrast with ristocetin and botrocetin, Polybrene did not cause increased aggregation of patient PRP. Polybrene aggregated normal washed platelets less extensively in the presence of patient plasma than normal plasma. These studies demonstrated that botrocetin induced heightened interaction between platelets and vWF, but Polybrene did not, in platelet-type vWD, and that the enhanced responsiveness of patient platelets to botrocetin is related to an intrinsic platelet abnormality.     

A new congenital platelet abnormality characterized by spontaneous platelet aggregation, enhanced von Willebrand factor platelet interaction, and the presence of all von Willebrand factor multimers in plasma

A case is reported of a 49-year-old woman with a mild bleeding tendency. Her bleeding time, platelet count and size, plasma ristocetin cofactor activity, von Willebrand factor (vWF) antigen, and vWF multimeric pattern are all within normal limits. Spontaneous platelet aggregation is observed when citrated platelet-rich plasma (PRP) is stirred in an aggregometer cuvette. This aggregation is completely is only slightly diminished by an antiglycoprotein (GP) IIb/IIIa or by an anti GPIb monoclonal antibody. The patient's PRP shows increased sensitivity to ristocetin. The distinct feature of this patient, also present in two family members studied, is that platelet aggregation is initiated by purified vWF in the absence of any other agonist. The vWF- induced platelet aggregation is abolished by anti-GPIb and anti- GPIIb/IIIa monoclonal antibodies and by EDTA (5 mmol/L). Apyrase inhibits the second wave of aggregation. Patient's platelets in PRP are four to six times more reactive to asialo vWF-induced platelet aggregation than normal platelets. The amount of radiolabeled vWF bound to platelets in the presence of either low concentration of ristocetin or asialo vWF was increased 30% compared with normal. The patient's platelet GPIb was analyzed by SDS page and immunoblotting and by binding studies with anti-GPIb monoclonal antibodies showed one band with slightly increased migration pattern and a normal number of GPIb molecules. Unlike the previously reported patients with pseudo or platelet-type von Willebrand disease, this patient has normal vWF parameters.     

Membrane fluidity and platelet aggregation: dibucaine permits ristocetin-induced platelet aggregation with low-molecular-weight von Willebrand multimers.

The effects of brief incubation of platelets with dibucaine were studied. Membrane fluidity was increased after incubation with 1 mM dibucaine for 1 min as determined by fluorescence polarization. Platelet aggregation was increased when ristocetin was employed as modulator and normal plasma used as a source of von Willebrand factor (vWF). In contrast, aggregation was decreased with botrocetin. After cryoprecipitation the supernatant, which contained predominantly low-molecular-weight vWF multimers, was effective with ristocetin only after prior incubation of the platelets with dibucaine. This indicates that low-molecular-weight vWF multimers can also support ristocetin-induced aggregation when the membrane fluidity is increased. This idea was supported by the use of plasma from a patient with von Willebrand disease type IIa. Being a multimeric protein, von Willebrand factor contains multiple binding sites for glycoprotein Ib (GP Ib). GP Ib-dependent aggregation by agents that do not contain multiple binding sites, i.e. wheat germ agglutinin or polyclonal antibodies to GP Ib, was decreased after brief incubation with dibucaine. These observations are discussed in relation to the hypothesis that the increased membrane fluidity allows an increased number of GP Ib molecules to bind to each vWF multimer.     

Spontaneous platelet aggregation during pregnancy in a patient with von Willebrand disease type IIB can be blocked by monoclonal antibodies to both platelet glycoproteins Ib and IIb/IIIa

Spontaneous platelet aggregation appeared in a patient with von Willebrand disease type IIB during the 37th week of pregnancy. This phenomenon was not associated with symptoms of thrombosis and the patient delivered by caesarean section with no complications. Her platelet-poor plasma (PPP) aggregated normal platelet-rich plasma (PRP) and washed platelets. Aggregation was inhibited by monoclonal antibodies with known specificity for the platelet receptors of von Willebrand factor (vWF), i.e. the glycoprotein Ib (GPIb) and the GPIIb/IIIa complex. A monoclonal antibody, which selectively inhibits the binding of vWF to the GPIIb/IIIa complex, did not block aggregation, suggesting that spontaneous aggregation is not dependent on the binding to GPIIb/IIIa of vWF from patient plasma. Aggregation induced by patient plasma could also be blocked either by two monoclonal antibodies raised against vWF or by a fragment derived from trypsin digestion of normal vWF which blocks the ristocetin-induced binding of normal vWF to platelets. These findings indicate that the spontaneous platelet aggregation in this patient results from the binding of her vWF to GPIb but is independent from the binding of her vWF to GPIIb/IIIa.     

Differential diagnosis of neonatal alloimmune thrombocytopenia: Type 2B von Willebrand disease

At birth, severe thrombocytopenia without context of infection should mainly suggest neonatal alloimmune thrombocytopenia (NAIT), especially in case of a platelet count below 20 GL^?1. We report two cases of severe neonatal thrombocytopenia, first suspected as being NAIT. Both had a platelet count below 20 GL^?1 with platelet clumps. The absence of alloantibodies and failure of platelet transfusion and intravenous immunoglobulins to improve the platelet count led to question the diagnosis and to evoke inherited bleeding disorders. Measurements of Von Willebrand factor (VWF) levels showed a marked reduction of VWF:RCo and a normal VWF:Ag, suggesting a type 2B Von Willebrand disease (VWD2B). Ristocetin-induced platelet aggregation could not be performed because of the very low platelet count. In the first case, after sequencing VWF exon 28, a heterozygous p.Leu1460Pro mutation was found consistent with VWD2B. In the second case, the genetic analysis of VWF exon 28 identified a homozygous mutation: p.Pro1337Leu confirming type VWD2B and also the p.Arg854Gln homozygous mutation in exon 20 confirming type 2N (ratio FVIII/VWF:Ag <0.5).

The two cases underline that, even if NAIT remains the most common diagnosis in severe neonatal thrombocytopenia, it should be challenged in the absence of documented incompatibility, chronic evolution, or treatment failure. Diagnosis of VWD2B should be considered in early thrombocytopenia, even without familial history. In the cases presented, genotyping confirmed the subtype of VWD and helped to guide the therapeutic management of bleeding episodes.     

Type IIB von Willebrand''s disease with probable autosomal recessive inheritance and presenting as thrombocytopenia in infancy

von Willebrand's disease (vWD) is a congenital bleeding disorder that exists in two main forms. In the classic form, type I, the concentration of the von Willebrand factor (vWF) in plasma is decreased. In type II vWD, the vWF is structurally altered. Type II can be further divided into at least six subtypes (A, B, C, D, E and F). In type IIB the vWF, in contrast to other variants of vWD, shows an increased affinity for platelets. IIB vWD is generally believed to be inherited in an autosomal dominant manner. We describe two families with three affected children in whom an autosomal recessive inheritance is more likely. Thrombocytopenia, constant or variable, was present from early infancy in all three cases. Type IIB vWD should thus be included in the differential diagnosis of congenital thrombocytopenia.     

A new von Willebrand variant (type I, New York): increased ristocetin-induced platelet aggregation and plasma von Willebrand factor containing the full range of multimers

We report three members of a family who had reduced levels of plasma von Willebrand factor (vWF) and increased ristocetin-induced platelet aggregation (RIPA) (aggregation of platelet-rich plasma with ristocetin at a concentration of 0.45 mg/mL), as previously reported in type IIB and pseudo-von Willebrand's disease (vWD). However, in contrast to the latter two disorders in which the larger vWF multimers are absent in plasma, the entire range of vWF multimers was observed in the patients' plasma after sodium dodecyl sulfate-agarose gel electrophoresis, and all vWF multimers (including the largest) were present in the same proportion as in normal plasma and type I vWD. Thus, despite increased RIPA, the levels and multimeric pattern of vWF in this family's plasma were indistinguishable from those in type I vWD in which RIPA is usually decreased. Addition of ristocetin to the patients' platelet- rich plasma resulted in the removal of vWF (and, more selectively, of the large multimers) at lower concentrations of ristocetin than normal, as in type IIB and pseudo-vWD. The defect in the patients was localized to their vWF, which had an enhanced capacity for aggregating washed normal platelets in the presence of low concentrations of ristocetin and for aggregating pseudo-vWD platelets (in the absence of ristocetin). Both glycoproteins (GP) Ib and IIb-IIIa were involved in the enhanced aggregation response. RIPA (at low ristocetin concentrations) in the patients' platelet-rich plasma was abolished by a monoclonal antibody (AP1) to GPIb and was markedly reduced by monoclonal antibodies (10E5 and LJP9) that block adenosine diphosphate and thrombin-induced binding of vWF and fibrinogen to GPIIb-IIIa but was unaffected by an antibody (LJP5) that only blocks vWF binding. Partial inhibition of the initial aggregation slope (and complete inhibition of second phase aggregation) was achieved with creatine phosphate/creatine phosphokinase. EDTA blocked second-phase aggregation but was without effect on the initial slope. The findings in this family combine some features of both type I vWD (normal pattern of vWF multimers in plasma) and type IIB vWD (increased RIPA) and further demonstrate the increasing complexity of the structure-function relationships in vWD.     

Studies of multimerin in patients with von Willebrand disease and platelet von Willebrand factor deficiency

In normal platelet α-granules von Willebrand factor (VWF) is stored with multimerin and factor V in an eccentric electron-lucent zone. Because the platelet stores of VWF are deficient in 'platelet low' type 1 and type 3 von Willebrand disease (VWD), we investigated their electron-lucent zone proteins. The patients with VWD had partial to complete deficiencies of plasma and platelet VWF but normal α-granular multimerin and factor V, and normal α-granular fibrinogen, thrombospondin-1, fibronectin, osteonectin and P-selectin. In type 3 VWD platelets, α-granular electron-lucent zones lacking VWF-associated tubules were identified and multimerin was found in its normal α-granular location. These findings indicate that the formation of the electron-lucent zone and the sorting of multimerin to this region occur independent of VWF. The isolated abnormalities in VWF suggests a VWF gene mutation is the cause of 'platelet low' type 1 VWD.     

Investigation of a large kindred with type IIB von Willebrand''s disease, dominant inheritance and age-dependent thrombocytopenia

Twenty-six members of four generations of one family in which a man was diagnosed in 1961 as having von Willebrand's disease (vWD) have been studied. Subtype IIB vWD and autosomal dominant inheritance was identified in 19 individuals with bleeding signs varying in severity and frequency. The absence of high molecular weight multimers of plasma von Willebrand factor (vWf) and the heightened interaction of plasma vWf with platelets in the presence of low ristocetin concentrations were consistent in all affected subjects. Nevertheless the degree of these abnormalities was variable without clearcut linkage to the severity of clinical symptoms. Thrombocytopenia was present only in the adult affected family members and the platelet count appeared to be age-dependent. The investigation of this family provides further evidence of the phenotypic variability in the vWf-platelet interactions within this vWD subtype.     

Pregnancy-induced worsening of thrombocytopenia in a patient with type IIB von Willebrand's disease.

Thrombocytopenia has been reported in patients with type IIB von Willebrand's disease (vWd) during pregnancy. In the present work we report the behaviour of platelet count and von Willebrand factor (vWf) multimers in a pregnant type IIB vWd patient who presented with mild thrombocytopenia in the steady state. The evolution of pregnancy was associated with a progressive decrease of platelet count which showed its lowest value two days before delivery (20 x 10(9)/l). The tendency of the platelet count to decrease was suddenly reversed a few days later (77 x 10(9)/l). At the same time, the vWf multimeric pattern showed a strict but inverse correlation with the platelet count. In fact, a progressive increase in low and intermediate sized vWf multimers, which proceeded until platelets reached their minimum level, was noted. A few days after delivery, concomitant with the prompt platelet increase, low and intermediate multimers became decreased. During pregnancy, the patient's platelet showed additional increased responsiveness to ristocetin but did not demonstrate spontaneous platelet aggregation (SPA). On the contrary, the patient's plasma, collected both during and after pregnancy, caused normal platelets to aggregate spontaneously. SPA appeared completely blocked by an anti-GPIIb-IIIa monoclonal antibody (MAb), which recognized the binding site for fibrinogen, vWf and fibronectin. In contrast, a MAb against ristocetin-induced vWf binding site on GPIb did not affect SPA. These findings suggest that the common stimulus or stimuli, responsible for the pregnancy-induced decrease of platelet count and improvement of vWf multimeric pattern in type IIB vWd is strictly related to pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)     

von Willebrand factor synthesized by endothelial cells from a patient with type IIB von Willebrand disease supports platelet adhesion normally but has an increased affinity for platelets.

Endothelial cells were isolated from the umbilical vein of a patient with subtype IIB von Willebrand disease, and the biosynthesis and function of von Willebrand factor (vWF) synthesized by these cells were compared with those of vWF synthesized by endothelial cells from normal individuals. The patient's endothelial cells synthesized, stored, and secreted vWF indistinguishably from normal endothelial cells: it was synthesized as a prepolypeptide of Mr 270,000 and had a mature form of Mr 220,000; the full spectrum of multimers was found both inside the cells and in the culture medium; it was stored normally, in the Weibel-Palade bodies; and similar amounts of vWF were secreted into the medium and deposited in the extracellular matrix. In a perfusion set-up, the extracellular matrix from IIB cells supported platelet adhesion similarly to the matrix from normal cells. vWF secreted constitutively by IIB cells into the culture medium bound to platelets at concentrations of ristocetin lower than those necessary for vWF from normal cells. vWF stored in the Weibel-Palade bodies of type IIB cells was released upon stimulation with phorbol ester and bound almost completely to platelets even in the absence of ristocetin. Moreover, spontaneous platelet aggregation was induced by vWF synthesized by type IIB cells. These data support the hypothesis that the absence of highly multimeric forms of vWF in plasma of type IIB von Willebrand disease patients is due to specific removal of these multimers by platelets.     

Cosegregation of von Willebrand factor gene polymorphisms and possible germinal mosaicism in type IIB von Willebrand disease

Recent reports of the mutations resulting in von Willebrand disease (vWD) have indicated that some cases of type IIA vWD are caused by single nucleotide substitutions in the gene encoding von Willebrand factor (vWF). However, the molecular pathogenesis of type IIB vWD remains unresolved and, with the complex posttranslational processing required for fully functional vWF, the mutations responsible for this phenotype may occur at loci other than the vWF gene. This study has used six intragenic vWF polymorphisms to assess the linkage of type IIB vWD to this gene in three families (48 individuals). The results of these studies indicate that there is significant linkage between the vWF gene and the type IIB phenotype (logarithm of the odds ratio of 7.2 at theta = 0), suggesting that the mutations responsible for this disorder frequently occur at this locus. Results from one of these families indicates that the disorder has been transmitted from an unaffected parent to two children who have inherited the same vWF gene as seven unaffected siblings. This finding is suggestive of the presence of germinal mosaicism for the mutation in the father.     

Identification of a point mutation in type IIB von Willebrand disease illustrating the regulation of von Willebrand factor affinity for the platelet membrane glycoprotein Ib-IX receptor.

von Willebrand factor (vWF) supports platelet adhesion on thrombogenic surfaces by binding to platelet membrane glycoprotein (GP) Ib in the GP Ib-IX receptor complex. This interaction is physiologically regulated so that it does not occur between circulating vWF and platelets but, rather, only at a site of vascular injury. The abnormal vWF found in type IIB von Willebrand disease, however, has a characteristically increased affinity for GP Ib and binds to circulating platelets. We have analyzed the molecular basis of this abnormality by sequence analysis of a type IIB vWF cDNA and have identified a single amino acid change, Trp550 to Cys550, located in the GP Ib-binding domain of the molecule comprising residues 449-728. Bacterial expression of recombinant fragments corresponding to this vWF domain yielded molecules that, whether containing a normal Trp550 or a mutant Cys550 residue, bound directly to GP Ib in the absence of modulators and with similar affinity. In contrast, mammalian cell expression of the same segment of sequence yielded molecules that, when containing the normal Trp550, did not bind to GP Ib directly but, like native vWF, bound in the presence of ristocetin. However, molecules containing the point mutation (Cys550) behaved like type IIB vWF--namely, bound to GP Ib even without ristocetin modulation and, in the presence of ristocetin, had 10-fold higher affinity than molecules with normal sequence. These results identify a region of vWF that, although not thought to be directly involved in binding to GP Ib, may modulate the interaction through conformational changes.     

Type IIB von Willebrand's disease associated with a complex thrombocytopenic thrombocytopathy

A familial bleeding disorder characterized by an association of Type IIB von Willebrand's disease (vWD) with a complex thrombocytopenic thrombocytopathy is described in two patients from the same generation. Findings typical of type IIB vWD included enhanced ristocetin-induced binding of patient von Willebrand factor (vWF) to platelets of patients and normal individuals in association with the absence of larger multimers from plasma. Abnormalities in platelet function included deficient platelet aggregation to ADP, collagen, epinephrine, and arachidonic acid; and defective release of 14C-serotonin, vWF, and platelet factor 4 (PF4) in response to thrombin, collagen, or ADP. Platelet factor 4 and platelet vWF were decreased when measured per mg of total platelet protein. In addition, the binding of normal vWF to patient platelets stimulated with thrombin was decreased. Platelet size was increased with a very heterogeneous distribution width. Electron microscopic evaluation showed giant platelets with dense and alpha bodies present. The platelet count was borderline or slightly decreased in the resting state and declined to frankly thrombocytopenic levels at the time of acute bleeding episodes; this state was associated with the presence of platelet aggregates in blood smears.     

A new variant of type II von Willebrand disease with aberrant multimeric structure of plasma but not platelet von Willebrand factor (type IIF)

A patient with a lifelong bleeding disorder was diagnosed as having Type II von Willebrand disease. The larger multimers of von Willebrand factor were absent from her plasma but present in platelets. A high- resolution electrophoretic technique was used to study the complex structure of individual von Willebrand factor multimers. In normal plasma, each multimer could be resolved into five bands: a more intense central one and four less intense, two moving faster and two slower than the central band. In normal platelets, each multimer could also be resolved into five bands. The central one had a mobility similar to that in plasma, whereas the four satellite bands had a mobility that differed from that of the corresponding plasma bands. In the patient, platelet von Willebrand factor antigen content and ristocetin cofactor activity were normal, and von Willebrand factor showed the same structure of individual multimers as seen in normal platelets. On the other hand, plasma von Willebrand factor antigen and ristocetin cofactor activity were decreased, and the structure of individual von Willebrand factor multimers was different from that of normal plasma and similar to that seen in normal and patient's platelets. After infusion of 1-deamino-8-D-arginine vasopressin, the largest von Willebrand factor multimers, as well as new satellite bands with a mobility similar to those in normal plasma, appeared in the patient plasma, and the levels of von Willebrand factor antigen and ristocetin cofactor activity became normal. Yet no relevant change in the prolonged bleeding time was observed. This new variant of von Willebrand disease, therefore, is characterized by the presence of a dysfunctional von Willebrand factor molecule that exhibits unique structural abnormalities in plasma but appears to be normal in platelets. The designation of Type IIF is proposed for this type of von Willebrand disease in accordance with the terminology that has been previously used.