Purification and characterization of human platelet von Willebrand factor

Summary. Platelet von Willebrand factor (vWf) was purified from human platelet concentrates. The multimeric structure of the purified platelet vWf was similar to that observed in the initial platelet lysate, and, like the platelet lysate, the purified platelet vWf contained higher molecular weight multimers than plasma vWf. The apparent molecular weight of the reduced platelet vWf subunit was similar to the plasma vWf subunit. The N-terminal amino acid of the purified platelet and plasma vWf was blocked. In concentration dependent binding to botrocetin- or ristocetin-stimulated platelets, ¹²⁵I-plasma vWf bound with a higher affinity than platelet. The ristocetin cofactor activity per mg of purified plasma vWf was 5-fold greater than the platelet vWf activity. Platelet and plasma vWf bound to collagen with similar affinities; however, platelet vWf bound to thrombin-stimulated platelets and to heparin with a higher affinity than plasma vWf. The differences in the binding affinity(s) of plasma and platelet vWf to platelet GPIb and GPIIb/IIIa and extracellular matrix proteins may reflect different roles for plasma and platelet vWf in the initial stages of haemostasis and thrombosis.     

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.     

Use of DDAVP in inherited and acquired platelet dysfunction

Twenty-one patients with prolonged bleeding times secondary to inherited disorders of platelet function and eight patients with prolonged bleeding times secondary to acquired platelet dysfunction were given 0.3 micrograms per kilogram of DDAVP, 1-deamino-8-D-arginine vasopressin, intravenously. Sixteen of twenty-two DDAVP trials in patients with inherited platelet dysfunction (73%) and seven of the nine DDAVP trials in patients with acquired platelet dysfunction (78%) resulted in normalization or shortening of the prolonged bleeding times by at least 4 min. The bleeding time response did not correlate with changes in the levels of von Willebrand factor (vWf) antigen or ristocetin cofactor activity, nor was it associated with changes in vWf multimeric analysis or in vitro platelet aggregations following the administration of DDAVP. Shortening of the bleeding time with DDAVP was seen in patients with a failure to release/storage pool type defect, thromboxane synthesis type defect, Bernard-Soulier syndrome, Glanzmann's thrombasthenia, the May-Hegglin anomaly, liver disease, nonuremic renal disease, myelofibrosis, and Tangier's disease.     

Shear-induced platelet aggregation increases in patients with proximal and severe coronary artery stenosis

BACKGROUND: Shear stress generated in stenosed arteries promotes platelet thrombi formation at the stenosed sites by accelerating the binding of von Willebrand factor (vWF) to platelets. Shear-induced platelet aggregation (SIPA) has been studied in acute coronary syndromes, but not in chronic coronary disease. HYPOTHESIS: We investigated the effect of both the site and severity of coronary stenosis on SIPA in patients with chronic coronary artery disease. METHODS: Shear-induced platelet aggregation was measured using platelet-rich plasma in 49 patients (41 men and 8 women; mean age 61+/-10 years) with coronary artery disease to evaluate the association between the extent of SIPA and coronary angiographic findings. Stenoses > 75% were considered severe. In all, 62 healthy individuals (54 men and 18 women; mean age 45+/-7 years) served as controls. The correlation between SIPA and the site and severity of the coronary lesion, and parameters of coagulation and fibrinolysis were evaluated. RESULTS: Shear-induced platelet aggregation was increased in the stenosis group (69.0+/-10.6%) compared with the controls (57.7+/-10.3%, p < 0.0001). Patients with severe stenosis in the proximal segments had significantly increased SIPA (p< 0.0001) and vWF larger multimer concentration (p<0.0001) compared with the control group. A significant correlation existed between SIPA and the vWF larger multimer concentration in all subjects studied (r = 0.422, p < 0.0001). CONCLUSIONS: Shear-induced platelet aggregation is increased in patients with severe stenosis of the proximal coronary arteries and correlates with plasma concentrations of vWF larger multimers, suggesting that severe stenosis in the proximal segments is not only associated with an increased risk of significant myocardial ischemia, but may also generate high shear stress in the stenosed artery and increase plasma vWF larger multimers, thereby promoting the formation of platelet thrombi.     

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.     

The molecular characterization of von Willebrand disease: good in parts

Since the cloning of the gene that encodes von Willebrand factor (VWF), 27 years ago, significant progress has been made in our understanding of the molecular basis of the most common inherited bleeding disorder, von Willebrand disease (VWD). The molecular pathology of this condition represents a range of genetic mechanisms, some of which are now very well characterized, and others that are still under investigation. In general, our knowledge of the molecular basis of type 2 and 3 VWD is now well advanced, and in some instances this information is being used to enhance clinical management. In contrast, our understanding of the molecular pathogenesis of the most common form of VWD, type 1 disease, is still at an early stage, with preliminary evidence that this phenotype involves a complex interplay between environmental factors and the influence of genetic variability both within and outside of the VWF locus.     

Multimeric pattern discrepancy between platelet and plasma von Willebrand factor in type IIC von Willebrand disease

von Willebrand factor (vWF) from platelet lysate and plasma, collected in the presence of protease inhibitors, was studied in two patients with type IIC von Willebrand disease (vWD). Platelet and plasma vWF showed the smallest multimer increased, but the latter had a repeating single band whereas the former had a repeating "doublet." This platelet-plasma discrepancy observed for the first time in these patients suggests that the repeating "doublet" or single band described in other type IIC patients represent minor subgroups of type IIC vWD.     

The role of the platelet function analyser (PFA-100TM) in the characterization of patients with von Willebrand''s disease and its relationships with von Willebrand factor and the ABO blood group

Determination of the closure time (CT) with the platelet function analyser (PFA-100) is a useful screening test for von Willebrand's disease (VWD) but its role in the characterization of VWD is not well established. We studied the relationship between the prolongation of the CT with adenosine diphosphate (ADP) (CT-ADP) and epinephrine (CT-EPI) cartridges and the von Willebrand factor (VWF) in 53 patients with VWD. We found that a relatively small percentage of the prolongation of the CT-ADR and CT-ADP (16 and 29%, respectively) was determined by a reduction in VWF levels. The CT-ADP was significantly more prolonged in the presence of qualitative defects in VWF but could not discriminate between the VWD subtypes. The ABO blood group had no effect on the prolongation of the CT or the bleeding time. In conclusion, the PFA-100 appears of little use in the characterization of severity and subtype of VWD.     

Role of ADP in platelet aggregation at high shear: studies in a patient with congenital defect of platelet responses to ADP

Summary. The in vitro measurement of platelet aggregation (PA) at the high shear levels that can be found in the microcirculation may provide useful informations on primary haemostasis, which is usually explored in vivo with the skin bleeding time (BT). PA at high shear requires von Willebrand factor (vWf) and the platelet glycoprotein (GP) complexes Ib/IX/V and IIb/IIIa; controversial results have been reported on its requirement of released adenosine diphosphate (ADP). Due to its dependence on vWf, PA at high shear may be affected by the vasopressin analogue DDAVP, which increases the plasma vWf levels and shortens the prolonged BT of patients with congenital or acquired defects of platelet function. We studied PA at high shear, BT and plasma vWf levels in a patient with congenital impairment of platelet responses to ADP before and after the i.v. infusion of 0.3 μg/kg DDAVP. Two methods to study PA at high shear were used: shear-induced PA (SIPA) and the filter aggregation test. With both methods, PA at high shear of the patient was impaired. The infusion of DDAVP increased plasma vWf levels, shortened the prolonged BT and potentiated PA at high shear of the patient. In conclusion, PA at high shear is impaired in a patient with congenital defect of platelet responses to ADP and prolonged BT and is potentiated by DDAVP. Our results suggest that released ADP plays an important role in PA at high shear and that potentiation of PA at high shear by DDAVP may be one mechanism by which the drug shortens the prolonged BT of patients with congenital or acquired defects of platelet function.     

DDAVP-induced enhancement of platelet retention: its dependence on platelet-von Willebrand factor and the platelet receptor GP IIb/IIIa.

Although DDAVP has been shown to be haemostatically efficacious in patients with various congenital or acquired platelet disorders, no reasonable explanation has been found for this effect. We have previously shown DDAVP to increase platelet adhesiveness as measured with a platelet retention test. The aim of the present study was to investigate the mechanism of action responsible for the increased platelet retention in response to DDAVP. Patients with vWD type III and type Ia, severe haemophilia and severe thrombasthenia, as well as healthy controls, were included in the study. The effect of different concentrations of vWF in plasma and platelets was explored, as was the effect on platelet function of apyrase and monoclonal antibodies against GP IIb/IIIa and GP Ib. We found the effect of DDAVP on platelet retention to be unaffected by changes in the plasma concentration of vWF. The enhanced platelet retention after DDAVP is apparently dependent on the presence of platelet-vWF and on a normal function of the GP IIb/IIIa. The effect is not mediated via ADP or thrombin. The platelet-stimulating effect of DDAVP may be one explanation for the positive haemostatic effect in patients with certain platelet disorders.     

A Leu7Pro mutation in the signal peptide of platelet glycoprotein (GP)IX in a case of Bernard-Soulier syndrome abolishes surface expression of the GPIb-V-IX complex

This paper describes the molecular defect of the second case of Bernard-Soulier syndrome, initially reported in 1957. Analysis of the patient's platelets by flow cytometry and Western blotting failed to detect surface expression of any of the four subunits of the glycoprotein (GP)Ib-V-IX complex and revealed small amounts of intracellular GPIbalpha, GPIbbeta and GPV but no GPIX. DNA sequencing revealed a novel missense mutation in the GPIX gene which replaced Leu (CTG) by Pro (CCG) at position 7 of the signal peptide. This mutation is, to date, the only known example of a leader sequence defect in Bernard-Soulier syndrome. The change occurred in a prototypic alpha-helical hydrophobic core region, typically enriched in leucine and devoid of proline residues. Co-transfection of GPIXPro7 with normal GPIbalpha and GPIbbeta into Chinese hamster ovary cells reproduced the platelet phenotype, resulting in no detectable GPIX, low intracellular levels of GPIbalpha and GPIbbeta, and an absence of surface expression. This mutation presumably leads to an abnormal conformation and, hence, incorrect insertion of GPIX into the endoplasmic reticulum and/or to defective signal peptide cleavage, both of which are required for correct transport to the cell membrane. This provides further evidence for a critical role of GPIX in controlling biosynthesis of the GPIb-IX complex.     

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.     

Decreased half-life time of plasma von Willebrand factor collagen binding activity in essential thrombocythaemia: normalization after cytoreduction of the increased platelet count

Patients with essential thrombocythaemia (ET) exhibit a decrease of large von Willebrand factor (VWF) multimers in plasma, which is inversely related to the platelet count. In the present study we investigated whether the decrease of large VWF multimers in plasma with increasing platelet counts is the consequence of increased turnover of large VWF multimers in vivo . To that end we measured the half-life times of endogenously released VWF:Ag and VWF:CBA (collagen binding activity) after intravenous administration of desmopressin (DDAVP) to nine ET patients and nine control subjects (N). In addition, the half-life times of VWF:Ag and VWF:CBA were also measured in four ET patients after cytoreduction of the increased platelet count to normal or nearly normal values. Estimated half-life times of VWF:Ag did not differ between ET patients and normals (11.0 ± 4.0 h v 12.4 ± 2.5 h, P   > 0.05). Estimated half-life times of VWF:CBA were significantly lower in ET patients as compared with normal individuals (6.1 ± 2.0 h v 8.4 ± 2.5 h, P   < 0.05). After cytoreduction of the increased platelet count to (nearly) normal values in all four ET patients the half-life time of VWF:CBA significantly ( P  = 0.014) increased from 5.2 ± 1.2 h to 8.7 ± 2.0 h. Our data suggest that platelets may play a role in the homeostasis of circulating von Willebrand factor, which may compromise normal haemostasis at fairly increased platelet counts.     

2型糖尿病合并大血管病变患者GMP-140、vWF检测的意义

目的：研究2型糖尿病患血小板膜糖蛋白－140（GMP－140）、血管性血友病因子（vWF)和血栓形成之间的关系，及与糖尿病合并大血管病变之间的联系。方法：采用酶联免疫吸附双抗体夹心法定量测定30名糖尿无并发症、35名糖尿病伴大血管病变患，35名正常人的血浆GMP－140、vWF水平。结果：2型糖尿病患的血浆GMP－140，vWF水平高于正常组（P＜0．01），糖尿病伴大血管病变的患上述变化更显（P＜0．001）。结论：2型糖尿病患血管内皮细胞损伤、血小板粘附性和聚集性增加，易于形成血栓，与大血管并发症有一定因果关系。     

A new mutation,S1285F,within the A1 loop of von Willebrand factor induces a conformational change in A1 loop with abnormal binding to platelet GPIb and botrocetin causing type 2M von Willebrand disease

We report the identification of a new mutation in exon 28 of the von Willebrand factor (VWF) gene in two related patients with type 2M von Willebrand disease (VWD). The molecular abnormality changes the Ser 1285 to Phe within the A1 loop of VWF. The S1285F mutation was reproduced by site-directed mutagenesis on the full-length VWF cDNA. The mutated recombinant VWF (rVWF), F1285rVWF, and the hybrid, S/F1285rVWF, were expressed in COS-7 cells. F1285rVWF exhibited a slight decrease of high-molecular-weight multimers and markedly reduced ristocetin- or botrocetin-induced binding of VWF to platelets in association with a decreased binding to botrocetin. The hybrid S/F1285rVWF showed a normal multimeric profile and bound to platelets in a similar way to the patients' plasma VWF, in the presence of ristocetin or botrocetin. Thus, the new S1285F mutation within the A1 loop was responsible for the type 2M VWD observed in these patients, and was involved in the binding of VWF to botrocetin and to platelet glycoprotein Ib (GPIb). Three anti-VWF monoclonal antibodies, with conformational epitopes within the A1 loop but distinct GPIb binding inhibitory properties, showed a different interaction with F1285rVWF. These results indicate that the S1285F substitution alters the folding of the A1 loop and prevents the correct exposure of the VWF binding sites to botrocetin and GPIb.     

Autosomal dominant C1149R von Willebrand disease: phenotypic findings and their implications

Background

Mutation C1149R in the von Willebrand factor (VWF) gene has been thought to cause autosomal dominant severe type 1 von Willebrand disease (VWD).

Design and Methods

Eight patients from three unrelated families with this mutation were included in the present study who had distinct VWF abnormalities, not described in earlier studies.

Results

The patients showed notably low levels of VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), VWF collagen binding (VWF:CB), and a reduced ristocetin-induced platelet aggregation (RIPA). VWF:RCo/VWF:Ag and VWF:CB/VWF:Ag ratios were lower than 0.7. At basal conditions, all the VWF multimers were decreased in plasma, with a clearly lower relative proportion of the high molecular weight VWF multimers (HMWM). In high-resolution agarose gels, a large decrease in the relative proportions of the satellite bands was seen. The patients had a brief good response to desmopressin (DDAVP) administration, but the released VWF half-life was shorter than normal, indicating an accelerated clearance of their VWF. Platelet VWF was abnormal.

Conclusions

We conclude from the results obtained in these patients for plasma phenotypic data that this mutation should be classified as a VWD type 2A (IIE). DDAVP therapy may be somewhat helpful for this mutation, at least for mild to moderate bleeding. These data provide evidence that for VWD classification factors other than basal VWF, such as DDAVP response and platelet VWF, should be considered.     

p38 MAPK is activated but not necessary in porcine von Willebrand factor-dependent platelet activation

We have investigated the role of p38 mitogen-activated protein kinase (MAPK) in von Willebrand factor (VWF)-dependent platelet activation. The interaction of platelets with subendothelial VWF, especially under high shear stress, is considered to be the first activation step which primes platelets for subsequent haemostatic events. As a model of VWF-dependent platelet activation, porcine VWF was employed. Porcine VWF induced p38 MAPK activation by 1 min post-addition; assessed by phosphorylation of a recombinant p38 MAPK fusion protein substrate termed glutathione S-transferase-MAPK activated protein kinase-2. To determine if p38 MAPK was necessary for porcine VWF-induced platelet activation, we functionally inhibited p38 MAPK activity with SB203580 before exposure of the platelets to porcine VWF. Inhibition of p38 MAPK had no effect on VWF-induced platelet alpha or lysozomal granule release, expression of activated GPIIb IIIa, modulation of membrane glycoprotein CD41, expression of phosphatidylserine as assessed by annexin V binding, microparticle formation, or platelet agglutination. It was concluded that SB203580-inhibitable p38 MAPK activity induced by porcine VWF is not necessary for platelet activation.     

Absence of incorporation of plasma von Willebrand factor into porcine platelet a-granules

Summary. In order to study the relationship between plasma and platelet von Willebrand factor (vWF), we used an experimental model of crossed bone marrow transplantation (BMT) between SLA immunocompatible normal and homozygous von Willebrand (vWD) pigs. A normal pig received bone marrow from a vWD pig and a second pig with vWD was engrafted with marrow from a normal pig.
Each recipient, after total irradiation of 10 Grays, received by a central catheter 10¹⁰ monocellular bone marrow cells without immunosuppression. The animals were followed for 50 d and no graft rejection or graft-versus-host disease was observed. After aplasia occurring 3 weeks after BMT, white blood cells and platelets returned to normal.
Before transplantation, in the vWD pig, vWFAg and vWF activity were not detected in plasma and in platelet and megakaryocyte a-granules. After transplantation with normal marrow, platelet vWFAg and platelet vWF activity were normal and high molecular weight multimers and numerous tubular structures were present in a-granules.
Before transplantation, the normal pig had normal plasma and platelet vWFAg-vWF activity, normal multimeric pattern, and the platelet and megakaryocyte a-granules displayed many tubular structures, eccentrically located in one of their poles, coinciding with immunogold staining vWFAg. After transplantation with homozygous vWD marrow, platelet and megakaryocyte a-granules lacked tubular structures. Alpha-granule immunogold staining for vWF was consistently negative, although plasma vWF was at a normal level. In conclusion, this study shows that, unlike other plasma proteins such as fibrinogen, vWF endocytosis does not occur from plasma to the platelet a -granules. Platelet and megakaryocyte vWF solely originates from megakaryocyte endogenous synthesis and is independent of plasma vWF.