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.     

Postpartum von Willebrand factor levels in women with and without von Willebrand disease and implications for prophylaxis

The aim of this study was to elucidate the fall in von Willebrand factor (VWF) and factor VIII activity (FVIII) after childbirth in women with and without von Willebrand disease (VWD). VWF:RCo, VWF:Ag, and FVIII were obtained in the third trimester of pregnancy, on admission for childbirth, and 10 times postpartum. Specimens were processed within 4 h and analysed centrally. Means were calculated at each time point. Forty women (40 pregnancies) without VWD and 32 women (35 pregnancies) with VWD were enrolled. 15/32 with VWD were treated (30% of those with type 1 and all of those with type 2) in 17 pregnancies. Treatments prior to delivery consisted of desmopressin (2/17), VWF concentrate (15/17) and after delivery VWF concentrate (16/17). Duration of treatment was 0–21 days (median 6). VWF levels peaked at 250% of baseline – 4 h postpartum in women with VWD and 12 h postpartum in women without VWD. Thereafter, VWF levels fell rapidly, approached baseline at 1 week and reached baseline at 3 weeks. Except immediately postpartum, when the levels among treated cases were higher, levels among women with VWD appeared to parallel, but were lower than those among women without VWD. Levels were lowest among those who received treatment. VWF levels fall rapidly after childbirth. Except immediately postpartum, current treatment strategies do not raise VWF levels to the levels of women without VWD or even to the levels of women with milder, untreated VWD. Consequently, women with VWD may be at risk of postpartum haemorrhage despite treatment.     

Unique interactions of asialo von Willebrand factor with platelets in platelet-type von Willebrand disease

The present studies demonstrate that platelets from patients with platelet-type von Willebrand disease show specific and saturable binding of asialo von Willebrand factor (AS-vWF) under conditions where such binding is not observed with normal platelets. Although specific binding of 125I-AS-vWF to formalin-fixed normal platelets could not be demonstrated, specific binding to fixed patient platelets was seen with an apparent Kd of 1.3 micrograms/mL and specific maximally bound ligand of 0.40 micrograms/10(8) platelets. Preincubation of patient platelets with the antiglycoprotein Ib (anti-GPIb) monoclonal antibody AS-2 reduced total binding close to the level of computer-estimated nonspecific binding. In contrast, binding was not reduced by preincubation with anti-GPIIb/IIIa monoclonal antibody or with 5 mmol/L EDTA. Under stirring conditions, the binding of AS-vWF to fixed patient platelets was accompanied by a strong agglutination response. AS-vWF- induced agglutination was similarly observed in patient but not normal platelet-rich plasma (PRP) in the presence of 5 mmol/L EDTA. In the absence of EDTA, AS-vWF produced a full aggregation response in patient PRP at concentrations as low as 0.1 microgram/mL in contrast to the 2 to 20 micrograms/mL required by normal PRP. Both thromboxane B2 formation and adenosine triphosphate secretion showed an AS-vWF concentration dependence paralleling the aggregation responses. These studies show that a major difference in the platelets from patients with platelet-type von Willebrand disease is the presence of an exposed, high-affinity binding site associated with GPIb that recognizes AS-vWF.     

Oligosaccharide structures of von Willebrand factor and their potential role in von Willebrand disease

Oligosaccharides make up approximately 20% of the mass of VWF and although their structures are well established, their functional role remains unclear. Modification of the VWF oligosaccharide structures has been shown to result in increased plasma clearance of the protein. A mutation which alters cell type-specific expression of the Galgt2 glycosyltransferase gene in the RIIIS/J mouse results in an autosomal dominant partial quantitative deficiency of VWF. Increased plasma clearance of VWF has been demonstrated in some individuals with a partial quantitative deficiency of the protein and it is possible that variation in VWF glycosylation may contribute towards this. ABH antigens occur within the oligosaccharide component of VWF and may account for the variation in plasma VWF:Ag levels observed between individuals of different ABO blood groups. The structures and functional roles of the oligosaccharide side chains of VWF and possible pathogenetic mechanisms by which they may contribute towards VWD are reviewed in this article.     

Insights into von Willebrand factor proteolysis: clinical implications

The proteolysis of von Willebrand factor (VWF) by the recently discovered metalloprotease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin repeats), is a normal processing step in VWF biochemistry. Emerging data indicate that this step may be influenced by a variety of factors, some of which favour increased proteolysis and some of which compromise proteolysis. The former may predispose to bleeding, whilst the latter appears to be the underlying mechanism for thrombotic thrombocytopenic purpura (TTP). The new insights support the concept of "risk" in bleeding, particularly in the case of type 1 von Willebrand disease (VWD), in much the same way that risk is considered in venous thrombosis. This review presents relevant current knowledge of VWF proteolysis by ADAMTS13, and a novel model of how this may be implicated in type 1 VWD is proposed, based on events at the vessel wall at a time of haemostatic challenge.     

von Willebrand factor in CHD and stroke: Relationships and therapeutic implications

Opinion statement Atherosclerotic cardiovascular disease (CVD), which includes coronary heart disease (CHD) and stroke, is now the most common cause of death in the middle aged and elderly in all parts of the world except subSaharan Africa. The direct cause of death is frequently an acute thrombotic arterial occlusion. Because atherosclerosis is a diffuse disease, patients with CHD also have a high risk of ischemic stroke. The hemostatic process is a needed defense mechanism to control hemorrhage after injury but at same time, if overactive, may have the potential to precipitate diseases such as myocardial infarction or stroke in the setting of atherosclerosis. In approximately 1% of all patients with ischemic stroke, and in up to 4% of young adults with stroke, the major precipitant of brain ischemia is a hematologic disorder or coagulopathy that predisposes to thrombosis. von Willebrand factor (vWF) plays an important role in platelet adhesion to subendothelial structures and in the intrinsic pathway of coagulation. It is regarded as an indirect measure of endothelial dysfunction. Deficiency of vWF in von Willebrand’s disease is well established. However, much less is known regarding the pathophysiologic implications of an elevated level of vWF, particularly in relation to CVD and cerebrovascular disease. The importance of vWF in the pathogenesis of this disease is poorly defined and information is limited and inconsistent. Elevated levels of vWF have been variably linked with risk of CHD; causal criteria are not fully met. Relationships with stroke risk are even less well established. Measurement of vWF adds little to risk prediction after considering the major risk factors—age, sex, smoking, raised blood cholesterol, and hypertension. vWF may have a greater role in predicting outcome in subjects with acute coronary syndromes (ACS), stroke, and perhaps atrial fibrillation. Investigation of the use of vWF level to guide treatment of ACS or stroke is ongoing; however, there is no compelling evidence to date.     

von Willebrand factor and platelet interactions with the vessel wall

von Willebrand factor (vWF) is a multimeric glycoprotein which has a dual role in haemostasis, functioning as carrier protein for Factor VIII and mediating platelet adhesion to exposed subendothelium (SE). vWF interacts with components of the SE such as collagen and heparin-like glycosaminoglycans as well as with two platelet membrane receptors: glycoprotein (GP) Ib and GPIIb/IIIa. These multiple binding functions explain its definition as an adhesive protein. vWF promotes platelet adhesion at the high shear rates which correspond to the rheologic conditions of the microcirculation or of narrowed arterial vessels. The role of the vWF-GPIb interaction in platelet adhesion is well known; that of the vWF-GPIIb/IIIa interaction has been more recently demonstrated through the use of monoclonal antibodies (MAbs) or synthetic peptides blocking vWF-binding to GPIIb/IIIa. In addition, perfusion studies in native, non-anticoagulated blood emphasize the concept that vWF is also essential for thrombus formation at high shear stress. Thus, vWF fragments, synthetic peptides or MAbs blocking the functional domains of vWF represent potential therapeutic strategies to prevent the development of thrombosis.     

Control of von Willebrand factor multimer size and implications for disease

Plasma von Willebrand factor (vWF) is a multimeric protein that mediates adhesion of platelets to sites of vascular injury, however only the very large vWF multimers are haemostatically competent. Plasma vWF derives predominantly from the vascular endothelium and is of a smaller average multimer size to that found in the sub-endothelial matrix. The existence of plasma factors that control the size of vWF multimers and account for this difference has long been suspected. vWF cleaving protease (vWFCP), a metalloproteinase that regulates vWF multimer size by proteolytic cleavage, and thrombospondin-1 (TSP-1), a trimeric glycoprotein that uncouples multimers by reducing the disulfide-bonds which link individual subunits, are two such factors that have recently been identified. The large and ultra large vWF multimers play a central role in arterial thrombosis and agents that regulate their size hold promise as novel anti-thrombotic drugs.     

Comparative analysis of type 2b von Willebrand disease mutations: implications for the mechanism of von Willebrand factor binding to platelets

von Willebrand factor (vWF) is a multimeric glycoprotein that forms an adhesive link following vascular injury between the vessel wall and its primary ligand on the platelet surface, glycoprotein Ib (GpIb). Type 2b von Willebrand disease (vWD) is a qualitative form of vWD resulting from enhanced binding of vWF to platelets. Molecular characterization of the vWF gene in patients with type 2b vWD has resulted in identification of a panel of mutations associated with this disorder, all clustered within the GpIb binding domain in exon 28 of the vWF gene. We have expressed six of the most common type 2b vWD mutations in recombinant vWF and show that each mutation produces a similar increase in vWF binding to platelets in the absence or presence of ristocetin. Furthermore, expression of more than one type 2b vWD mutation in the same molecule (cis) or in different molecules within the same multimer (trans) failed to produce an increase in vWF platelet binding compared with any of the individually expressed mutations. Taken together, these data support the hypothesis that the vWF GpIb binding domain can adopt either a discrete "on" or "off" conformation, with most type 2b vWD mutations resulting in vWF locked in the on conformation. This model may have relevance to other adhesive proteins containing type A domains.     

The role of von Willebrand factor in platelet function

von Willebrand factor (vWF) has essential functions in the very first stage of hemostasis. At the site of vascular damage vWF binds immediately to exposed collagens, thereby facilitating the adhesion of platelets. After a first layer of platelets has been formed, vWF is also crucial as a link between platelets in the forming thrombus. In this article, the current knowledge on the role of vWF in primary hemostasis is reviewed.     

Platelet--von Willebrand factor interactions in type IIB von Willebrand's disease

Type IIB von Willebrand's disease (vWD) is a distinct form of this disorder in which the largest multimers of the von Willebrand factor (vWF) are lacking in plasma but present in platelets. When the vasopressin analogue, 1-deamino-8-D-arginine vasopressin (DDAVP), is given to patients with type IIB vWD, an abnormal vWF is released to plasma. This vWF causes thrombocytopenia in vivo and platelet aggregation in vitro. Aggregation occurs in the plasma milieu and thus at physiological fibrinogen concentration. In this study we demonstrate that IIB post-DDAVP vWF aggregated only metabolically active platelets. The platelet aggregation was completely inhibited by EDTA and PGE1, and either inhibited or greatly weakened by ASA, demonstrating the role of divalent cations and thromboxane A2 formation. In spite of inhibiting platelet aggregation, EDTA, PGE1 and ASA did not prevent platelet binding of IIB post-DDAVP vWF. An antiserum against GP Ib made normal platelets less responsive to the IIB vWF although neither platelet aggregation nor vWF binding were completely prevented. The aggregation was fibrinogen-dependent and platelets from patients with Glanzmann's thrombasthenia were unresponsive. The studies provide evidence that IIB post-DDAVP vWF is bound to unstimulated platelets and that the interaction between vWF and platelets in type IIB vWD is different from ristocetin-induced as well as thrombin- and epinephrine-induced binding to platelets of normal vWF.     

Biosynthesis, processing and secretion of von Willebrand factor: biological implications.

von Willebrand factor is a multimeric plasma glycoprotein that is required for normal haemostasis. von Willebrand factor is synthesized by endothelial cells and megakaryocytes, and originates from its precursor pro-von Willebrand factor. The endoproteolytic processing of pro-von Willebrand factor results in mature von Willebrand factor and von Willebrand factor propeptide (also known as von Willebrand Ag II). In endothelial cells, the propeptide controls the polymerization and subsequent targeting of von Willebrand factor to the storage vesicles, the so-called Weibel-Palade bodies. Upon stimulation of the endothelial cells, the Weibel-Palade bodies are translocated to the plasma membrane of the cell, and mature von Willebrand factor and its propeptide are co-secreted. After release, these polypeptides have divergent fates and serve different biological functions. Mature von Willebrand factor both controls platelet adhesion and aggregation at sites of vascular injury and acts as a chaperone protein for coagulation factor VIII. The von Willebrand factor propeptide may serve a role in modulating inflammatory processes. This still growing body of information indicates that the biological function of the von Willebrand factor gene product is more diverse than was previously thought.     

Inhibitor development in haemophilia A: the role of von Willebrand factor/factor VIII concentrates

Summary.  The presence of inhibitors that neutralize the function of factor VIII (FVIII) decreases the haemostatic efficacy of replacement clotting factor concentrate and increases morbidity among patients with haemophilia A. Certain genetic and environmental variables have been linked to a higher incidence of inhibitors. Conversely, the presence of von Willebrand factor (VWF) in some plasma-derived FVIII products may provide some measure of protection against inhibitor development, although the evidence is not conclusive. Clinical trials are needed to resolve this issue and determine the appropriate role of VWF-containing FVIII concentrates in the treatment of haemophilia A patients.     

Sickle cell vasoocclusion and rescue in a microfluidic device

The pathophysiology of sickle cell disease is complicated by the multiscale processes that link the molecular genotype to the organismal phenotype: hemoglobin polymerization occurring in milliseconds, microscopic cellular sickling in a few seconds or less [Eaton WA, Hofrichter J (1990) Adv Protein Chem 40:63-279], and macroscopic vessel occlusion over a time scale of minutes, the last of which is necessary for a crisis [Bunn HF (1997) N Engl J Med 337:762-769]. Using a minimal but robust artificial microfluidic environment, we show that it is possible to evoke, control, and inhibit the collective vasoocclusive or jamming event in sickle cell disease. We use a combination of geometric, physical, chemical, and biological means to quantify the phase space for the onset of a jamming event, as well as its dissolution, and find that oxygen-dependent sickle hemoglobin polymerization and melting alone are sufficient to recreate jamming and rescue. We further show that a key source of the heterogeneity in occlusion arises from the slow collective jamming of a confined, flowing suspension of soft cells that change their morphology and rheology relatively quickly. Finally, we quantify and investigate the effects of small-molecule inhibitors of polymerization and therapeutic red blood cell exchange on this dynamical process. Our experimental study integrates the dynamics of collective processes associated with occlusion at the molecular, polymer, cellular, and tissue level; lays the foundation for a quantitative understanding of the rate-limiting processes; and provides a potential tool for optimizing and individualizing treatment, and identifying new therapies.     

Investigation of the role of von Willebrand factor in thrombotic thrombocytopenic purpura

Von Willebrand factor (vWF) has been implicated to function as a cofactor in platelet aggregation induced by thrombotic thrombocytopenic purpura (TTP) plasma. To investigate further this role of vWF, we have used rabbit monospecific anti-FVIII/vWF antibodies and a monoclonal antibody to platelet glycoprotein Ib (GP Ib) that blocks the ristocetin- induced platelet aggregation. The monoclonal anti-platelet GP Ib antibody inhibited the platelet aggregation induced by ristocetin in the presence of normal plasma, but not that by any of the five TTP plasma samples. The TTP plasma samples from five patients were incubated with the monospecific antibodies to FVIII/vWF. In all of the samples, the FVIII/vWF:Ag was drastically reduced; however, there was almost no effect on the platelet-aggregating activity. Therefore, it is concluded that vWF is unlikely to play a major role in platelet aggregation induced by majority of TTP plasmas and that the site of platelet GP Ib, to which vWF binds in the presence of ristocetin, is not involved in TTP plasma-induced aggregation.     

Screening for von Willebrand disease: contribution of an automated assay for von Willebrand factor activity

Measuring von Willebrand factor (VWF) activity is essential to the diagnosis of von Willebrand disease (VWD). The VWF activity is usually assessed based on measurement of the ristocetin cofactor (VWF:RCo). However, that test is technically challenging and has high intra- and inter-assay variabilities. The HemosIL VWF activity (VWF:AC) is a fully automated assay, recently proposed as a good alternative to VWF:RCo for VWD diagnosis. This study was undertaken to assess this new method. First, the analytical performance of VWF:AC on an automated coagulo-meter (ACLTop) was determined, and then this new method was compared with VWF:RCo and the platelet function analyzer (PFA100) for 160 patients referred for VWD screening. The VWF:AC achieved acceptable precision with within-run and between-run coefficients of variation ranging from 2.3% to 14.1%, and linearity from 10% to 100%. Despite some marked differences between VWF:AC and VWF:RCo for 10 plasmas tested, their agreement for VWD diagnosis was good. The VWF:AC had sensitivity similar to that of PFA100 (close to 100%), but better specificity (97.7% vs. 66% or 60%, depending on the cartridge used). The good analytical performance, and the sensitivity and specificity of VWF:AC to detect VWF deficiency renders it a suitable method for VWD screening. Our findings support VWF:AC use for the diagnostic work-up of VWD, paying close attention to concomitant clinical signs and bleeding score, as recommended for VWD.     

Platelet – von Willebrand factor interactions in Type IIB von Willebrand's disease

Type IIB von Willebrand's disease (vWD) is a distinct form of this disorder in which the largest multimers of the von Willebrand factor (vWF) are lacking in plasma but present in platelets. When the vasopressin analogue, l-deamino-8-D-arginine vasopressin (DDAVP), is given to patients with type IIB vWD, an abnormal vWF is released to plasma. This vWF causes thrombocytopenia in vivo and platelet aggregation in vitro. Aggregation occurs in the plasma milieu and thus at physiological fibrinogen concentration. In this study we demonstrate that IIB post-DDAVP vWF aggregated only metabolically active platelets. The platelet aggregation was completely inhibited by EDTA and PGE₁, and either inhibited or greatly weakened by ASA, demonstrating the role of divalent cations and thromboxane A₂ formation. In spite of inhibiting platelet aggregation, EDTA, PGE₁ and ASA did not prevent platelet binding of IIB post-DDAVP vWF. An antiserum against GP Ib made normal platelets less responsive to the IIB vWF although neither platelet aggregation nor vWF binding were completely prevented. The aggregation was fibrinogen-dependent and platelets from patients with Glanzmann's thrombasthenia were unresponsive. The studies provide evidence that IIB post-DDAVP vWF is bound to unstimulated platelets and that the interaction between vWF and platelets in type IIB vWD is different from ristocetin-induced as well as thrombin- and epinephrine-induced binding to platelets of normal vWF.     

Proteolytic processing of von Willebrand factor subunit: Heterogeneity in type-IIA von Willebrand disease

Summary Type IIA von Willebrand disease (vWD) is a heterogeneous disorder for which two different pathogenetic mechanisms have been proposed: increased proteolytic susceptibility of von Willebrand factor (vWF), and/or interference of its post-translational processing. Subunit analysis of vWF in type-IIA vWD has revealed an increased relative proportion of the 176- and 140-kDa subunit-derived fragments, suggesting an augmented fragmentation of vWF, even in the resting state. We analyzed the subunit pattern of vWF in plasma from five previously described patients with type-IIA vWD. All of them showed the above-mentioned pattern. In addition, the presence of a new band with an apparent molecular mass of 200 kDa, not described in normal individuals or in patients with vWD, was repeatedly observed in one of these patients. This patient also exhibited an abnormal vWF multimeric structure in platelets and in plasma, before and after desmopressin administration, when the blood was collected either in the presence or in the absence of proteinase inhibitors. We believe that an abnormal primary structure of vWF could be responsible for this abnormal proteolytic fragmentation pattern, as well as for the abnormal multimerization of vWF. Moreover, an abnormal susceptibility to proteolysis appears to be present, as suggested by the increase in the relative proportion of the 176-kDa fragment observed in the same patient. Future sequencing studies and genetic analysis may clarify whether there are one or two different defects related to the vWF of that patient. Our results indicate that the subunit analysis of vWF may reveal additional defects present in type-IIA vWD that may help our understanding of the pathogenesis of such disease.Supported in part by grants 90/3229, 91-92/0372, 94/1509 (FIS, INSALUD, Spain).     

Increased factor VIII/von Willebrand factor antigen and von Willebrand factor activity in renal failure.

Factor VIII/von Willebrand factor antigen and von Willebrand factor activity (ristocetin assay) were studied in 12 patients in renal failure. A dramatic increase in both activities was observed (antigen 315 +/- 30 per cent in patients verus 104 +/- 9 per cent in control subjects; activity 402 +/- 48 per cent in patients versus 111 +/- 5 per cent in control subjects; p less than 0.001 for both). Since von Willebrand factor is thought to play at least a facilitative role in the development of arteriosclerosis, these increased activities may contribute to the premature arteriosclerosis reported in patients with chronic renal failure undergoing dialysis.