The role of platelet von Willebrand factor in platelet adhesion and thrombus formation: a study of 34 patients with various subtypes of type I von Willebrand disease

Summary In order to investigate the respective role of plasma and platelet von Willebrand factor (vWF) in mediating platelet adhesion and thrombus formation, we performed ex vivo perfusion studies with native blood from patients with various subtypes of type I von Willebrand disease (vWD). We studied 34 patients with type I vWD (19 'platelet normal', five 'platelet low', two 'platelet discordant', eight 'Vicenza'). Parallel studies were carried out on nine patients with severe vWD (type III). At high shear rate (2600 s^-1) we found that the defect in platelet-vessel wall interactions in patients having a normal platelet vWF content ('platelet normal' and 'Vicenza') involved thrombus formation, whereas platelet adhesion was normal. At this high shear rate, platelet adhesion and thrombus volume were significantly decreased in patients with subtypes 'platelet low' and 'platelet discordant', i.e. when platelet vWF is either low or dysfunctional. These results indicate that platelet vWF may substitute for plasma vWF to promote platelet adhesion. emphasizing the important role of platelet vWF. They also confirm the role of vWF in thrombus formation at high shear rate because an abnormal thrombus volume was observed in all patients. even when platelet adhesion was normal.     

Hemostatic effect of platelet von Willebrand factor.

In type III von Willebrand disease (vWD) patients, the bleeding time was only partially corrected or not modified after cryoprecipitate infusion, although the levels and the multimeric structure of plasma von Willebrand factor (vWF) were normal. However, the adhesion of normal platelets on the vessel wall subendothelium in the presence of postinfusion patient plasma improved more significantly than the bleeding time. These results suggest a role of the vWF released from normal platelets which is absent in type III vWD platelets. In 5 patients transfusion of normal platelet concentrates performed 1 h after cryoprecipitate infusion without modification of the bleeding time (> 30 min) normalized this parameter, and platelet adhesion to the subendothelium elicited a marked improvement. These last results confirm the suggestion that platelet vWF plays an important 'in vivo' role in the hemostatic process, particularly in patients suffering from severe vWD.     

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.     

Platelet adhesion to fibronectin in flow: the importance of von Willebrand factor and glycoprotein Ib

We describe glycoprotein (GP) Ib as a mediator of adhesion to fibronectin, specifically in flow. A monoclonal antibody (MoAb) directed to the von Willebrand factor (vWF)-binding site on this receptor or the absence of this receptor on the platelet membrane, in the case of a patient with the Bernard-Soulier syndrome, reduced platelet coverage to fibronectin to approximately 30% of the control value. A MoAb directed to the GP Ib-binding site on vWF showed a similar effect. With washed platelets in the absence of plasma vWF, the inhibitory effect of the anti-GP Ib antibody was the same as with whole blood. No inhibition with the anti-GP Ib antibody was observed when we used blood from patients with severe von Willebrand disease (vWD) or from a patient with vWD type I (platelet low). Addition of vWF to vWD blood resulted in restoration of adhesion. Immunoelectron microscopy on platelets adhering to fibronectin showed that GP Ib was homogeneously distributed over the entire surface of the platelet. vWF was present at the central zone and the edges of the platelet and at the basal interface between the platelet and the fibronectin surface. No direct binding of vWF to fibronectin could be demonstrated. These data indicate that GP Ib-mediated adhesion to fibronectin fully depends on vWF and that normal levels of plasma or platelet vWF are sufficient for optimal adhesion to fibronectin. The data suggest that the presence of platelets during perfusion is a prerequisite for vWF to support platelet adhesion to fibronectin.     

Platelet von Willebrand factor: evidence for its involvement in platelet adhesion to collagen

Although it is well established that plasma von Willebrand Factor (vWF) is essential to platelet adhesion to subendothelium at high shear rates, the role of platelet vWF is less clear. We studied the respective role of both plasma and platelet vWF in mediating platelet adhesion to fibrillar collagen in a parallel-plate perfusion chamber. Reconstituted blood containing RBCs, various mixtures of labeled washed platelets and plasma from controls or five patients with severe von Willebrand disease (vWD), was perfused through the chamber for five minutes at a shear rate of 1,600 s-1. Platelet-collagen interactions were estimated by counting the radioactivity in deposited platelets and by quantitative morphometry. When the perfusate consisted of normal platelets suspended in normal plasma, platelet deposition on the collagen was 24.7 +/- 3.6 X 10(6)/cm2 (mean +/- SEM, n = 6). Significantly less deposition (16 +/- 2.3) was observed when vWD platelets were substituted for normal platelets. In mixtures containing vWD plasma, significantly greater deposition (9 +/- 2.2) was obtained with normal than with vWD platelets (1 +/- 0.4) demonstrating a role for platelet vWF in mediating the deposition of platelets on collagen. Morphometric analysis confirmed these data. Our findings indicate that platelet, as well as plasma, vWF mediates platelet-collagen interactions at a high shear rate.     

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.     

Molecular diagosis of von Willebrand disease

von Willebrand disease (vWD) is one of the most common inherited human bleeding disorders, which is caused by quantitative or qualitative defects of von Willebrand factor (vWF). vWF is a highly multimerized glycoprotein that promotes platelet adhesion and aggregation at a high shear rate, while also acting as a carrier of coagulation factor VIII. vWD has been subdivided into three categories, which reflect their pathophysiology. Type 1 and type 3 vWD reflect partial or complete deficiency of vWF, whereas type 2 vWD reflects qualitative defect of vWF. The ability of vWF to interact with its platelet receptor and factor VIII, and the analysis of the multimeric composition of vWF are essential to identify patients with different vWD subtypes. The prevalence of different vWD subtypes was reported in the literature. In the past years, ninety-one patients with vWD were consulted in our institution. Of all the vWD patients, 56 (61.5%) belong to type 1, 26 (28.6%) type 2 and 9 (9.89%) type 3. The analysis of vWF gene was performed in some type 2 and type 3 vWD by denature gradient gel electrophoresis and sequencing. We have found six cases of point mutations of vWF gene, Ala737-->Glu, Gly 22-->Glu, Met37 Val and Ser71-->stop codon. Substitutions, are first reported in international database. We constructed an expression plasmid pSVA737EvWF containing full length of cDNA of vWF which included the Ala737 Glu substitution by site-direct mutagenesis. The structure of recombinant vWF within transfected COS-7 cells and the secretion of high-molecular-weight (HMW) multimers were similar to wild-type vWF. HMW forms of vWF multimers were absent in plasma but present in platelets. The mutation corresponds to the group II type 2A vWD characterized by normal secretion of all vWF multimers.     

Role for platelet von Willebrand factor in supporting platelet-vessel wall interactions in von Willebrand disease

Twelve infusions of plasma concentrates of von Willebrand factor (vWF) were given to four patients with severe (type III) von Willebrand disease (vWD). Their prolonged bleeding times were either completely or partially corrected after five infusions and had not changed after the remaining seven. In contrast, the low platelet coverage of the subendothelial surface of rabbit aorta perfused with normal washed platelets and red cells resuspended in preinfusion patient plasma was completely or partially corrected in ten instances by replacing preinfusion plasma with postinfusion plasma and remained unchanged in two. Postinfusion improvement in surface coverage was greater than that in bleeding time, suggesting that vWF from normal platelets is needed to support optimal platelet-vessel wall interactions in vWD. This possibility was further explored through other perfusion experiments. The subendothelial surface covered by platelets from an untreated patient with type III vWD (containing no measurable vWF) or from a type IIA vWD patient (containing dysfunctional vWF) resuspended in normal plasma was much smaller than that covered by normal platelets resuspended in normal plasma. These results establish that platelet vWF is important in supporting platelet-vessel wall interactions in vWD and also provide experimental support in favour of the therapeutic transfusion of normal platelets in addition to vWF concentrates to correct the bleeding time in vWD patients.     

Diagnosis of von Willebrand disease

Summary. von Willebrand disease (vWD) is a bleeding disorder caused by quantitative or qualitative defects of von Willebrand factor (vWF). vWF is synthesized by endothelial cells and megakaryocytes and circulates in plasma as a multimeric high molecular weight glycoprotein. vWF plays a major role in the early phases of ostasis by promoting platelet-vessel wall and plateletplatelet interactions under high shear conditions. It is also the carrier of coagulation factor VIII (FVIII) in plasma. A deficiency of vWF results in impairment of both primary and secondary phases of ostasis. Therefore, patients with vWD manifest bleeding symptoms that are typical of defects of primary ostasis (mucocutaneous haemorrhages) but, in case of severe deficiency of vWF, there are also haemarthroses and haematomas, which are typical of those seen with coagulation defects. Several types and subtypes of vWD have been described with a high degree of heterogeneity. The diagnosis is 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. Due to the heterogeneity of vWF defects, a correct diagnosis of types and subtypes may be sometimes difficult but is very important for an appropriate treatment of patients with vWD.     

New variant of von Willebrand disease type II with markedly increased levels of von Willebrand factor antigen and dominant mode of inheritance: von Willebrand disease type IIC Miami

A variant of von Willebrand disease (vWD) was identified in six members of a kindred spanning four generations. The proband was a 46-year-old woman with a lifelong history of bleeding, a prolonged bleeding time (> 15 minutes), markedly elevated von Willebrand factor (vWF) antigen (vWF:Ag = 2.09 U/mL), slightly reduced ristocetin cofactor activity, and a plasma vWF multimer pattern similar to that of vWD type IIC. Similar findings were observed in her three children, mother, and brother. In affected family members, platelet and plasma vWF multimer patterns were discrepant with higher molecular weight multimers observed in platelet vWF. Following a 1-Des-amino-8-D-arginine vasopressin (DDAVP) challenge, the proband failed to normalize her bleeding time even though vWF: Ag rose by 70% and higher molecular weight multimers were increased slightly. Genetic studies were consistent with autosomal dominant inheritance of a mutation within the vWF gene. By sequencing of cloned genomic DNA, mutations were excluded in exons 4, 5, 14, and 15, which encode regions of the vWF propeptide proposed to be important in multimer biosynthesis. Mutations also were excluded in exons 28 to 31, which encompass the known mutations that cause vWD types IIA, IIB, and B. This new variant of vWD, characterized by autosomal dominant inheritance, a qualitative defect that resembles vWD type IIC, and increased plasma vWF:Ag, was tentatively designated vWD type IIC Miami.     

Function of von Willebrand factor after crossed bone marrow transplantation between normal and von Willebrand disease pigs: effect on arterial thrombosis in chimeras.

von Willebrand factor (vWF) is essential for the induction of occlusive thrombosis in stenosed and injured pig arteries and for normal hemostasis. To separate the relative contribution of plasma and platelet vWF to arterial thrombosis, we produced chimeric normal and von Willebrand disease pigs by crossed bone marrow transplantation; von Willebrand disease (vWD) pigs were engrafted with normal pig bone marrow and normal pigs were engrafted with vWD bone marrow. Thrombosis developed in the chimeric normal pigs that showed normal levels of plasma vWF and an absence of platelet vWF; but no thrombosis occurred in the chimeric vWD pigs that demonstrated normal platelet vWF and an absence of plasma vWF. The ear bleeding times of the chimeric pigs were partially corrected by endogenous plasma vWF but not by platelet vWF. Our animal model demonstrated that vWF in the plasma compartment is essential for the development of arterial thrombosis and that it also contributes to the maintenance of bleeding time and hemostasis.     

Diagnosis and management of von Willebrand disease

von Willebrand disease (vWD) is a bleeding disorder caused by quantitative or qualitative defects of von Willebrand factor (vWF). The diagnosis is based on measurements of plasma and platelet vWF, the ability of vWF to interact with its platelet receptor and the analysis of the mutlimeric composition of vWF. Due to the heterogeneity of vWF defects, a correct diagnosis of types and subtypes may be sometimes difficult but is very important for an appropriate therapy. The aim of treatment is to correct the dual defects of haemostasis, i.e. abnormal coagulation expressed by low levels of factor VIII (FVIII) and abnormal platelet adhesion expressed by a prolonged bleeding time (BT). Desmopressin is the treatment of choice in patients with type 1 vWD, who account for approximately 80% of cases, because it corrects the FVIII/vWF levels and the prolonged BT in most of these patients. In type 3 and in the majority of type 2 vWD patients, desmopressin is not effective and it is necessary to resort to plasma concen-trates containing FVIII and vWF. Treated with virucidal methods, these concentrates are effective and currently safe, but the BT defect is not always corrected by them. Platelet concentrates or desmopressin can be used as adjunctive treatments when poor correction of the BT after concentrates is associated with continued bleeding.     

Laboratory monitoring of therapy in von Willebrand disease: efficacy of the PFA-100 and von Willebrand factor:collagen-binding activity as coupled strategies

Clinical management of von Willebrand disease (or von Willebrand disorder [vWD]) often involves factor replacement or desmopressin acetate (DDAVP) therapy to control (potential) bleeding. Laboratory monitoring involves testing patient samples prior to therapy and at discreet time points after therapy. Classical testing generally comprises assays for factor VIII:coagulant activity, von Willebrand factor (vWF):antigen and vWF:ristocetin cofactor activity. The PFA-100 (platelet function analyser) is a relatively new tool for the investigation of primary hemostasis, and studies have shown its potential utility in identifying both vWD and platelet disorders, and in monitoring DDAVP therapy in these patients. However, the PFA-100 has limited utility in monitoring factor replacement therapy. The collagen-binding activity (vWF:CB) assay is a relatively new functional vWF assay and studies have also shown its utility in identifying vWD, and in monitoring both DDAVP and factor replacement therapy in these patients. This review assesses the laboratory monitoring of therapy for vWD with a special focus on the combined potential utility of the PFA-100 and a vWF:CB assay sensitive for the presence or absence of large vWF multimers. This review should be of value to both hemostasis scientists and clinical specialists.     

Functional Property of von Willebrand Factor Under Flowing Blood

von Willebrand factor (vWF) is produced in megakaryocytes and endothelial cells, is stored in the alpha-granule of platelets and in the Weibel-Palade body of endothelial cells, and is present in plasma and vascular subendothelium. This huge protein with a unique multimeric structure plays a pivotal role in both hemostasis and pathological intravascular thrombosis, in which vWF contributes to both platelet adhesion/aggregation and blood coagulation through its multiple adhesive functions for the platelet membrane receptors, glycoprotein Ib-IX-V complex, integrin alphaIIbbeta3, heparin, various types of collagen, and coagulation factor VIII. Among various functions, the most characteristic feature of vWF is its determinant role on platelet thrombus formation under high-shear-rate conditions. Indeed, at in vivo rheological situations where platelets are flowing with high speed in the bloodstream, the only reaction that can initiate mural thrombogenesis is the interaction of vWF with platelet glycoprotein Ibalpha. The recent x-ray analysis of the crystal structure of various functional domains and functional studies of this protein under experimental flow conditions have rapidly advanced and revised our knowledge of the structure-function relationships of vWF, a key protein for hemostasis and arterial thrombosis.     

The roles of von Willebrand factor and factor VIII in arterial thrombosis: studies in canine von Willebrand disease and hemophilia A

We have studied the roles of von Willebrand factor (vWF) and factor VIII in arterial thrombosis in four canine phenotypes: normal (n = 6), hemophilia A (n = 11), von Willebrand disease (vWD) (n = 9), and hemophilia A/vWD (n = 1). vWF activity was determined by botrocetin- induced agglutination of fixed human platelets and vWF antigen (vWF:Ag) by Laurell electroimmunoassay and crossed immunoelectrophoresis. Plasma from normal dogs and those with hemophilia A had vWF activity, vWF:Ag, and a full range of vWF:Ag multimers on gel electrophoresis equivalent to normal canine plasma pool. Platelet cytosol contents were isolated by freezing and thawing, triton X-100 solubilization, or sonication of washed platelets with and without protease inhibitors and inhibitors of platelet activation. Washed platelets were also stimulated with calcium ionophore and MgCl2. There was no measurable vWF activity or vWF:Ag in platelet lysates or releasates in any dog regardless of phenotype. All dogs were studied using a standard arterial stenosis and injury procedure to induce arterial thrombosis. Thromboses were detected by cyclic reductions in Doppler blood flow velocity. Vessels were examined by light and scanning electron microscopy. Thrombosis developed in the arteries of normal (9 of 10) and hemophilia A dogs (16 of 16) but in none of the vWD dogs (0 of 10). Infusion of canine vWF cryoprecipitate into vWD dogs markedly shortened bleeding time but did not support thrombosis as seen in dogs with vWF in the plasma and subendothelium. Thrombosis, then, fails to occur when vWF is absent from the plasma and subendothelial compartments or present only in the plasma compartment. These data are consistent with the hypothesis that vWF in the plasma and subendothelium supports thrombosis. Neither plasma FVIII nor platelet vWF is essential for thrombosis in this model.     

Laboratory identification of von Willebrand disease: technical and scientific perspectives

The correct diagnosis and classification of von Willebrand disease (von Willebrand disorder; vWD) is crucial because the presenting biological activity of von Willebrand factor (vWF) determines both the hemorrhagic risk and subsequent clinical management. Many laboratory assays are employed, given that assay limitations and vWD heterogeneity results in no single test being able detect all forms of vWD. Minimal laboratory identification requires assessments of vWF:antigen, factor (F) VIII:coagulant activity, and functional vWF (using vWF:ristocetin cofactor activity and vWF:collagen-binding activity). Tests to help subclassify vWD include ristocetin-induced platelet aggregation, vWF:multimers, and vWF:FVIII binding assays. New diagnostic developments are now influencing vWD diagnosis, including advancements in methodologies, automation, new platelet function analyzers, genetic mutational analysis, and a better understanding of therapeutic pharmacokinetics. This review focuses on the current recommended laboratory process for investigation of vWD from a practical scientific technical laboratory perspective. Selection of appropriate combination test panels and testing sequence is crucial for the proper diagnosis and classification of congenital vWD.     

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.     

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.     

Factor VIII/von Willebrand factor in subendothelium mediates platelet adhesion

The present studies were undertaken to determine whether factor VIII/von Willebrand factor (vWF) present in the vessel wall (in addition to that in plasma) may mediate the attachment of platelets to subendothelium. Subendothelium from everted rabbit aorta was exposed to human citrated blood flowing through an annular perfusion chamber at 40 mL/min (wall shear rate of 2,600 s-1 for five minutes). The vessel segments were incubated at 37 degrees C for one hour with various dilutions of either goat-anti-rabbit factor VIII/vWF serum or an IgG fraction prepared from the serum. Control segments were incubated with serum or IgG from a nonimmunized goat. Values of platelet contact (C), platelet adhesion (C + S), and thrombus formation (T) on the subendothelium were evaluated by a morphometric technique. Compared with vessels incubated with fractions prepared from a normal goat, a significant decrease in platelet adhesion (C + S), ranging from 45% to 65%, was observed on vessels incubated with various dilutions (1:5 to 1:50) of either serum or IgG fractions of goat-anti-rabbit factor VIII/vWF. A similar decrease in platelet adhesion was observed with vessels incubated with an F(ab')2 fragment against rabbit factor VIII/vWF prepared in the goat. When goat-anti-rabbit factor VIII/vWF IgG was added to rabbit blood (1:75 dilution), platelet adhesion was reduced to the same extent (65%) on normal rabbit vessels and on vessels pre-incubated with goat-anti-rabbit factor VIII/vWF. Immunofluorescence studies revealed the presence of rabbit factor VIII/vWF in the subendothelium of rabbit aorta and the continued binding of the goat-anti-factor VIII/vWF antibodies on subendothelium during the perfusion studies. No uptake of human factor VIII/vWF on the rabbit subendothelium was observed by this immunologic technique; human factor VIII/vWF was found to be entirely associated with the attached human platelets. Thus, factor VIII/vWF in the vessel wall may mediate platelet attachment to subendothelium in a manner similar to that of plasma factor VIII/vWF.