ADAMTS13 and Von Willebrand factor in patients undergoing hemodialysis

Hemodialysis (HD) is associated with increasing thrombotic trend. Vascular access thrombosis (VAT) increases morbidity in HD patients. The aim of this study was to evaluate ADAMTS13 and VWF plasma levels from patients undergoing HD as putative biomarkers of the hypercoagulability state, as well the association between these markers and VAT occurrence. This study included 195 patients on HD for more than 6 months. HD patients were allocated into two groups according to the occurrence or not of previous episode of VAT; HD with VAT (N = 46) and HD without VAT (N = 149). ADAMTS13 and VWF were performed by ELISA. There was no significant difference between HD patients with and without VAT for ADAMTS13 and VWF levels. However, VWF levels were higher (P < 0.001) and ADAMTS13 were lower (P < 0.001) in HD patients, comparing to the control group composed by healthy subjects without kidney disease, age and sex-matched (N = 80). Taken together our data suggest a potential role of the kidneys function compromised on ADAMTS13 synthesis or metabolism, regardless other known sources of ADAMTS13. The imbalance between ADAMTS13 and VWF levels does not explain the development of VAT in HD patients by itself, although it should contribute for the hypercoagulability state. Therefore, additional studies to identify other risk factors are warranted and essential for better management of HD patients.     

The relationship between ABO groups and subgroups, factor VIII and von Willebrand factor

The aim of this study was to correlate ABO groups with plasma levels of factor VIII (FVIII), von Willebrand factor (VWF:Ag), and ristocetin cofactor (VWF:RCo). Serological and molecular tests defined blood groups from 114 donors (10 AA, 10 BB, 10 AB, 10 AO1, 10 BO1,16 O1O1, 20 A2O1, 20 A2B, 4 A3O1, 3 AxO1, and 1 BelO1). The levels of VWF:Ag, FVIII and VWF:RCo observed in rare subgroups (A3O1, AxO1, BelO1) were similar to the values found in the O1O1 group. However, levels of these factors were significantly higher in A2O1 donors than in O1O1 donors (VWF:Ag p=0.01; FVIII p=0.04; VWF:RCo p<0.001). Strong correlations were demonstrated between plasma levels of VWF:Ag and FVIII (R=0.77; p=0.001) and between VWF:Ag and VWF:RCo (R=0.75; p=0.001).     

Factor VIII accelerates proteolytic cleavage of von Willebrand factor by ADAMTS13

Proteolytic processing of von Willebrand factor (VWF) by ADAMTS13 metalloproteinase is crucial for normal hemostasis. In vitro, cleavage of VWF by ADAMTS13 is slow even at high shear stress and is typically studied in the presence of denaturants. We now show that, under shear stress and at physiological pH and ionic strength, coagulation factor VIII (FVIII) accelerates, by a factor of approximately 10, the rate of specific cleavage at the Tyr(1605)-Met(1606) bond in VWF. Multimer analysis reveals that FVIII preferentially accelerates the cleavage of high-molecular-weight multimers. This rate enhancement is not observed with VWF predenatured with 1.5 M guanidine. The ability of FVIII to enhance VWF cleavage by ADAMTS13 is rapidly lost after pretreatment of FVIII with thrombin. A FVIII derivative lacking most of the B domain behaves equivalently to full-length FVIII. In contrast, a derivative lacking both the B domain and the acidic region a3 that contributes to the high-affinity interaction of FVIII with VWF exhibits a greatly reduced ability to enhance VWF cleavage. Our data suggest that FVIII plays a role in regulating proteolytic processing of VWF by ADAMTS13 under shear stress, which depends on the high-affinity interaction between FVIII and its carrier protein, VWF.     

ADAMTS13 content in plasma‐derived factor VIII/von Willebrand factor concentrates

Thrombotic thrombocytopenic purpura (TTP) is a microangiopathy syndrome caused by a congenital or acquired deficiency of ADAMTS13, a plasma metalloprotease that cleaves von Willebrand factor (VWF) and thus prevents the formation of platelet‐rich thrombi in the microcirculation. TTP can be fatal if not appropriately and timely treated with the infusion of fresh frozen plasma (FFP) or exchange plasmapheresis, that reverse the process of microangiopathy by removing anti‐ADAMTS13 autoantibodies and replacing functional ADAMTS13. The treatment of TTP with FFP is not free from risks and must be administered in hospitals or clinics, owing to the substantial amount of plasma volume infused or exchanged and the frequent need of catheter application. Moreover, most FFPs are not subjected to treatments to remove or inactivate blood‐borne infectious agents. A number of recent reports indicate that certain plasma‐derived VWF‐factor VIII (FVIII) concentrates are clinically effective in the treatment of congenital TTP. In this study, we measured ADAMTS13 levels in various plasma‐derived VWF‐FVIII concentrates, showing that Koate^®‐DVI (Grifols), contained relatively high amounts of ADAMTS13 and that Alphanate^® (Grifols) was the closest other product in terms of protease content. Koate^®‐DVI contains, on average (five lots tested), 0.091 ± 0.007 Units of ADAMTS13 activity per IU of FVIII. On the basis of this analysis and other reports of VWF‐FVIII concentrate utilization in congenital TTP, potential dosing, and future clinical developments are discussed. Am. J. Hematol. 88:895–898, 2013. © 2013 Wiley Periodicals, Inc.     

Relationship between ABO and Secretor genotype with plasma levels of factor VIII and von Willebrand factor in thrombosis patients and control individuals

In contrast to earlier reports, this study examined the relationship between plasma levels of factor VIII (FVIII) and von Willebrand factor (VWF) and ABO blood group and secretor status at the genetic level in 355 patients with venous thrombosis as well as in 236 controls. ABO glycosyl transferase alleles A(1) and B were more frequent in the thrombosis collective and alleles O(1), O(2) and A(2) were more frequent in the controls. A low-risk group for venous thrombosis of individuals with genotypes O(1)O(1), O(1)O(2) and O(1)A(2) (H-antigen rich) could be distinguished from a high-risk group with genotypes A(1)A(1), A(1)B, O(1)A(1) and O(1)B (H-antigen poor). In both the thrombosis and control groups, the H-antigen rich group showed significantly lower levels of FVIII coagulant activity (FVIII:C) and VWF antigen (VWF:Ag) than the H-antigen poor group. The frequency of the different secretor genotypes in the thrombosis group was not different from that in the control group. No significant differences of FVIII:C and VWF:Ag levels were seen between SeSe, Sese and sese individuals in the thrombosis and in the control group. Thus the risk of venous thrombosis is associated with the ABO blood group genotype but not with secretor status.     

Allosteric activation of ADAMTS13 by von Willebrand factor

The metalloprotease ADAMTS13 cleaves von Willebrand factor (VWF) within endovascular platelet aggregates, and ADAMTS13 deficiency causes fatal microvascular thrombosis. The proximal metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains of ADAMTS13 recognize a cryptic site in VWF that is exposed by tensile force. Another seven T and two complement C1r/C1s, sea urchin epidermal growth factor, and bone morphogenetic protein (CUB) domains of uncertain function are C-terminal to the MDTCS domains. We find that the distal T8-CUB2 domains markedly inhibit substrate cleavage, and binding of VWF or monoclonal antibodies to distal ADAMTS13 domains relieves this autoinhibition. Small angle X-ray scattering data indicate that distal T-CUB domains interact with proximal MDTCS domains. Thus, ADAMTS13 is regulated by substrate-induced allosteric activation, which may optimize VWF cleavage under fluid shear stress in vivo. Distal domains of other ADAMTS proteases may have similar allosteric properties.After vascular injury, platelets adhere to von Willebrand factor (VWF) multimers bound to endothelial cell surfaces and connective tissue. The force of flowing blood on a growing platelet-rich thrombus stretches the central A2 domain of VWF and exposes a Tyr¹⁶⁰⁵-Met¹⁶⁰⁶ cleavage site for ADAMTS13 (Fig. 1A) (1–5), a metalloprotease that severs VWF and releases adherent platelets. Deficiency of ADAMTS13 disrupts this feedback regulatory mechanism and causes thrombotic thrombocytopenic purpura (TTP), which is characterized by life-threatening microvascular thrombosis (3, 6, 7).Open in a separate windowFig. 1.Activation of ADAMTS13 by autoantibodies from a patient with TTP or by low pH. (A) Structure of ADAMTS13. (B) Fluorogenic substrates terminate at VWF residues indicated by arrows. Each substrate has Lys¹⁶¹⁷ replaced with Arg, N-terminal Gly modified with IRDye QC-1 (QC1), and Asn¹⁶¹⁰ replaced by Cys and modified with DyLight 633 (DyL) (22). The arrowhead indicates the cleaved Tyr-Met bond. Secondary structure elements of the VWF A2 domain (11) are indicated below and segments that interact with specific ADAMTS13 domains (13) are indicated above the sequence. (C) BCW49 plasma activated ADAMTS13 with a titer of 9.6 U at pH 7.4 (orange squares), but not at pH 6.0 (orange circle). BCW49 plasma did not activate MDTCS at pH 6 (blue circle) or pH 7.4 (blue circle). (D) Rates of VWF71 cleavage were determined as a function of pH for ADAMTS13 (orange circles) and MDTCS (blue circles). Error bars indicate 95% confidence intervals and if not shown are smaller than the symbols.The recognition and cleavage of VWF is a formidable challenge. VWF and ADAMTS13 occur at ∼10 µg/mL and ∼1 µg/mL, respectively, compared with total plasma protein of ∼80,000 µg/mL. ADAMTS13 is constitutively active and has no known inhibitors in vivo. Nevertheless, VWF is the only identified ADAMTS13 substrate, and VWF is resistant to cleavage until subjected to fluid shear stress (8), adsorbed on a surface (9), or treated with denaturants (8, 10). This specificity depends on structural features of both ADAMTS13 and VWF that have not been characterized fully.The proximal metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains domains of ADAMTS13 bind to cryptic sites that are uncovered by unfolding VWF domain A2 (11-15) (Fig. 1B), and these interactions are required for efficient cleavage of VWF or peptide substrates. More distal ADAMTS13 domains bind to sites in or near VWF domain D4 that are always available (16–18). Deletion of distal ADAMTS13 domains impairs the cleavage of VWF multimers in vitro (16, 19) and increases VWF-dependent microvascular thrombosis in vivo (20) but accelerates the cleavage of peptide substrates (12, 13). In addition, ADAMTS13 cleaves guanidine hydrochloride-treated VWF multimers with an apparent K_m of ∼15 nM (21), which is 100-fold lower than the K_m of ∼1.6–1.7 µM for peptide substrates that are based on the sequence of VWF domain A2 (12, 14). These striking differences suggest that distal T or complement c1r/c1s, sea urchin epidermal growth factor, and bone morphogenetic protein (CUB) domains regulate ADAMTS13 activity. We have now shown that these distal domains inhibit ADAMTS13, and binding to VWF relieves this autoinhibition.     

Interplay between ADAMTS13 and von Willebrand factor in inherited and acquired thrombotic microangiopathies

The presence of unusually large multimers of von Willebrand factor (VWF) is thought to be a major pathogenic factor for thrombotic thrombocytopenic purpura (TTP). ADAMTS13 is a protease that regulates the multimeric size and function of VWF by cleaving VWF. Hence, congenital or acquired deficiency of ADAMTS13 causes life-threatening illness of TTP. Mutations in the ADAMTS13 gene cause inherited TTP, and the development of autoantibodies that inhibit ADAMTS13 activity frequently are associated with acquired TTP. ADAMTS13 consists of 1,427 amino acid residues and is composed of multiple structural and functional domains, containing a signal peptide, a propeptide, a reprolysin-like metalloprotease domain, a disintegrin-like domain, a thrombospondin type-1 (Tsp1) motif, a cysteine-rich domain, a spacer domain, seven additional Tsp1 repeats, and two CUB domains. In particular, the cysteine-rich/spacer domains are essential for VWF cleavage and are the principal epitopes recognized by autoantibodies in patients with acquired TTP. Therefore, it is likely that these domains are involved in the recognition and binding of ADAMTS13 to VWF. ADAMTS13 circulates in the blood in an active state, and efficiently cleaves unfold form of VWF induced under shear stress caused by blood flow, preventing the accumulation of pathogenic unusually large VWF multimers (ULVWF). Thus, ADAMTS13 helps maintain vascular homeostasis by preventing the excess thrombus formation.     

Bleeding time, blood groups and von Willebrand factor

The bleeding time in healthy volunteers was determined according to both the Ivy and the Simplate II techniques. A significantly longer bleeding time in people with blood group O than in people with non-O blood groups was demonstrated with both techniques. This difference could not be attributed to a difference in sex ratio, platelet count or haematocrit. The mean level of von Willebrand factor in blood group O is lower than in non-O blood groups, but we found no association between the level of von Willebrand factor and the bleeding time, despite the very broad range of von Willebrand factor levels in the subjects examined.     

von Willebrand factor cleaving protease and ADAMTS13 mutations in childhood TTP

Thrombotic thrombocytopenic purpura (TTP) is caused by the persistence of the highly reactive high-molecular-weight multimers of von Willebrand factor (VWF) due to deficiency of the specific VWF-cleaving protease (VWF-CP) ADAMTS13, resulting in microangiopathic disease. The acquired form is caused by autoantibodies against VWF-CP, whereas homozygous or compound heterozygous mutations of ADAMTS13 are responsible for recessively inherited TTP. We investigated 83 children with hemolytic or thrombocytopenic episodes with or without additional neurologic symptoms or renal failure. The presumed diagnosis was chronic idiopathic thrombocytopenic purpura (ITP; n = 50), TTP (n = 8), hemolytic uremic syndrome (HUS; n = 24), and Evans syndrome (n = 1). A severe deficiency of VWF-CP (< or = 5%) was found in all investigated patients with TTP and in none of those with HUS. Additionally, 2 of 50 patients with a prior diagnosis of ITP were deficient for VWF-CP. Antibodies against VWF-CP were found in 4 children. Mutation analysis of the ADAMTS13 gene in the patients deficient in VWF-CP by direct sequencing of all 29 exons identified 8 different mutations, suggesting the hereditary form of TTP in 1 patient with ITP, in the patient with Evans syndrome, and in 5 of the 8 patients with TTP. The phenotype of TTP in childhood can be rather variable. Besides the classical clinical picture, oligosymptomatic forms may occur that can delay the identification of patients at risk.     

Correlation between von Willebrand factor antigen,von Willebrand factor ristocetin cofactor activity and factor VIII activity in plasma

Background The laboratory diagnosis of von Willebrand Factor (VWF) deficiencies includes qualitative and quantitative measurements of VWF and clotting factor VIII (FVIII). Since the FVIII activity is frequently normal in patients with mild type 1 or 2 von Willebrand disease (VWD), there is controversy whether FVIII testing should accompany VWF Antigen (VWF:Ag) assay. Methods The aim of this study was to explore the correlation between VWF:Ag, VWF ristocetin cofactor activity (VWF:RCo) and FVIII in 213 consecutive patients undergoing screening for VWD. Results Forty-six patients were identified with VWF:Ag levels lower than the diagnostic threshold (54 IU/dl). A significant correlation was observed between VWF:Ag and VWF:RCo (r = 0.892; p < 0.001), VWF:Ag and FVIII (r = 0.834; p < 0.001), VWF:RCo and FVIII (r = 0.758; p < 0.001). Receiver operating characteristic curve analysis of the VWF:Ag assay revealed an area under the curve of 0.978 and 0.957 for detecting life-threatening values of FVIII (<30 IU/dl) and VWF:RCo (<40 IU/dl), respectively. The negative and positive predictive values at the VWF:Ag threshold value of 54 IU/dl were 100% and 33% for detecting life-threatening FVIII deficiencies, 94% and 80% for identifying abnormal values of VWF:RCo. Conclusions Due to the excellent correlation between VWF:Ag and FVIII and to the diagnostic efficiency of VWF:Ag for identifying abnormal FVIII levels in patients with VWF deficiency, routine measurement of FVIII may not be necessary in the initial screening of patients with suspected VWD. However, the limited negative predictive value of VWF:Ag for identifying type 2 VWD does not allow to eliminate VWF:RCo or VWF:FVIIIB assays from the diagnostic workout.     

Inflammation-associated ADAMTS13 deficiency promotes formation of ultra-large von Willebrand factor

In a prospective, longitudinal study, we investigated the association between decreased ADAMTS13 activity and impaired hemostasis, as well as organ dysfunctions in patients with systemic inflammation due to extracorporeal cardiopulmonary circuit or with severe sepsis. Similar to negative acute phase proteins, ADAMTS13 activity declined stepwise according to the extent of inflammatory responses. A marked imbalance between ADAMTS13 activity and VWF antigen level was associated with the appearance of ultra-large VWF multimers in plasma, with organ dysfunction and lethality. Our data support the view that systemic inflammation results in an ADAMTS13 deficiency which activates hemostasis.     

ADAMTS13 and von Willebrand factor and the risk of myocardial infarction in men

Von Willebrand factor (VWF) mediates the tethering/adhesion of platelets at sites of vascular injury. This function depends on its multimeric size, which is controlled by ADAMTS13. We measured plasma ADAMTS13 and VWF antigen levels by enzyme-linked immunosorbent assay (ELISA) in a large population-based case-control study (Study of Myocardial Infarctions Leiden [SMILE]), consisting of 560 men with a first myocardial infarction (MI) and 646 control subjects. Although ABO blood groups influenced VWF levels, they had no influence on ADAMTS13. Furthermore, there was no relationship between plasma ADAMTS13 and VWF levels. Similar to VWF, the estimated risk of MI was increased for every quartile of ADAMTS13 when compared to the lowest quartile (odds ratio, 1.5-1.6). If confirmed, the association of ADAMTS13 with MI may suggest an unexpected mechanistic action of ADAMTS13.     

Von Willebrand factor, ADAMTS13, and thrombotic thrombocytopenic purpura

Discoveries during the past decade have revolutionized our understanding of idiopathic thrombotic thrombocytopenic purpura (TTP). Most cases in adults are caused by acquired autoantibodies that inhibit ADAMTS13, a metalloprotease that cleaves von Willebrand factor within nascent platelet-rich thrombi to prevent hemolysis, thrombocytopenia, and tissue infarction. Although approximately 80% of patients respond to plasma exchange, which removes autoantibody and replenishes ADAMTS13, one third to one half of survivors develop refractory or relapsing disease. Intensive immunosuppressive therapy with rituximab appears to be effective as salvage therapy, and ongoing clinical trials should determine whether adjuvant rituximab with plasma exchange also is beneficial at first diagnosis. A major unanswered question is whether plasma exchange is effective for the subset of patients with idiopathic TTP who do not have severe ADAMTS13 deficiency.     

N-Glycans of ADAMTS13 modulate its secretion and von Willebrand factor cleaving activity

Severe deficiency of ADAMTS13, a plasma metalloprotease, leads to thrombotic thrombocytopenic purpura. ADAMTS13 contains 10 putative N-glycosylation sites in or near its metalloprotease sequence, spacer region, thrombospondin type 1 repeat no. 4 (TSR no. 4), and CUB domains. Tunicamycin treatment markedly decreased the secretion of ADAMTS13 into the culture medium of transfected cells. Nevertheless, the protease was efficiently secreted from N-acetylglucosaminyltransferase I-deficient Lec1 Chinese hamster ovary cells, indicating that N-glycosylation in the endoplasmic reticulum, but not the conversion of oligomannose to complex N-glycans in the Golgi complex, is important for secretion. However, ADAMTS13 with oligomannose N-glycans cleaved its substrate, von Willebrand factor (VWF) multimers, less effectively, with a higher K(m) but similar k(cat) value. In mutagenesis analysis, decreased secretion and VWF cleaving activity was observed with the N146Q and N828Q mutants, while decreased secretion only was observed with the N552Q mutant of ADAMTS13. Enzymatic removal of N-glycans from ADAMTS13 did not affect its VWF cleaving activity. Thus, N-glycosylation is necessary for efficient secretion of ADAMTS13, while conversion of the N-glycans from oligomannose to complex type in the Golgi complex enhances the proteolytic activity of the protease toward VWF multimers. After its secretion, ADAMTS13 does not require N-glycans for its VWF cleaving activity.     

Extensive contacts between ADAMTS13 exosites and von Willebrand factor domain A2 contribute to substrate specificity

The metalloprotease ADAMTS13 efficiently cleaves only the Tyr(1605)-Met(1606) bond in the central A2 domain of multimeric von Willebrand factor (VWF), even though VWF constitutes only 0.02% of plasma proteins. This remarkable specificity depends in part on binding of the noncatalytic ADAMTS13 spacer domain to the C-terminal alpha-helix of VWF domain A2. By kinetic analysis of recombinant ADAMTS13 constructs, we show that the first thrombospondin-1, Cys-rich, and spacer domains of ADAMTS13 interact with segments of VWF domain A2 between Gln(1624) and Arg(1668), and together these exosite interactions increase the rate of substrate cleavage by at least approximately 300-fold. Internal deletion of Gln(1624)-Arg(1641) minimally affected the rate of cleavage, indicating that ADAMTS13 does not require a specific distance between the scissile bond and auxiliary substrate binding sites. Smaller deletions of the P2-P9 or the P4'-P18' residues on either side of the Tyr(1605)-Met(1606) bond abolished cleavage, indicating that the metalloprotease domain interacts with additional residues flanking the cleavage site. Thus, specific recognition of VWF depends on cooperative, modular contacts between several ADAMTS13 domains and discrete segments of VWF domain A2.     

Prognostic value of plasma von Willebrand factor and its cleaving protease ADAMTS13 in patients with atrial fibrillation

Background

The von Willebrand factor (vWF) is essential for platelet adhesion and arterial thrombosis. It is degraded into less active multimers by ADAMTS13. Patients with atrial fibrillation (AF) exhibit higher plasma vWF and lower ADAMTS13 antigen levels. The vWF/ADAMTS13-ratio might help to estimate the pro-thrombotic risk of patients with AF. We therefore investigated whether a high ratio of vWF/ADAMTS13, independently of clinical risk scores, predicts major adverse cardiovascular events (MACE) in patients with AF.

Methods

This prospective longitudinal single center study included 269 patients with AF. Blood samples were analyzed for vWF and ADAMTS13-antigen concentration by means of enzyme-linked immunoassay kits.

Results

After adjustment for all univariable predictors for MACE (p ≤ 0.1), ADAMTS13 ≤ 49.77% (HR 1.833 (95% CI 1.089–3.086); p = 0.023) and vWF/ADAMTS13-ratio > 27.57 (HR 2.174 (95% CI 1.238–3.817); p = 0.007) remained independently associated with outcome. vWF > 1434.92 mU/ml (HR 1.539 (95% CI 0.883–2.682); p = 0.128) alone failed to independently predict MACE. In patients with low and intermediate risk for MACE according to the CHADS₂-score the addition of high vWF/ADAMTS13-ratio levels (> 27.57) had significant impact on the patients' outcome.

Conclusion

A high ratio of vWF/ADAMTS13 independently predicts MACE in patients with AF. Therefore, vWF and its cleaving protease ADAMTS13 might play an important role in the development and perpetuation of vascular disease in AF patients. This might be a novel target for future treatment strategies or an additional help for risk stratification in AF patients.     

Unraveling the scissile bond: how ADAMTS13 recognizes and cleaves von Willebrand factor

von Willebrand factor (VWF) is a large adhesive glycoprotein with established functions in hemostasis. It serves as a carrier for factor VIII and acts as a vascular damage sensor by attracting platelets to sites of vessel injury. VWF size is important for this latter function, with larger multimers being more hemostatically active. Functional imbalance in multimer size can variously cause microvascular thrombosis or bleeding. The regulation of VWF multimeric size and platelet-tethering function is carried out by ADAMTS13, a plasma metalloprotease that is constitutively active. Unusually, protease activity of ADAMTS13 is controlled not by natural inhibitors but by conformational changes in its substrate, which are induced when VWF is subject to elevated rheologic shear forces. This transforms VWF from a globular to an elongated protein. This conformational transformation unfolds the VWF A2 domain and reveals cryptic exosites as well as the scissile bond. To enable VWF proteolysis, ADAMTS13 makes multiple interactions that bring the protease to the substrate and position it to engage with the cleavage site as this becomes exposed by shear. This article reviews recent literature on the interaction between these 2 multidomain proteins and provides a summary model to explain proteolytic regulation of VWF by ADAMTS13.     

Size regulation of von Willebrand factor-mediated platelet thrombi by ADAMTS13 in flowing blood

The metalloproteinase ADAMTS13 regulates the size of released von Willebrand factor (VWF) multimers bound to endothelial cells, but it is unknown whether it can cleave plasma VWF during thrombogenesis. To address this issue, we perfused blood over immobilized VWF and used videomicroscopy to visualize an activation-independent platelet aggregation process mediated by soluble VWF at shear rates greater than 10 000 s(-1). At normal Ca2+ concentration, platelets formed rolling as well as surface-attached clusters that grew larger during the first 5 minutes but then lost more than 70% of their mass by 10 minutes. In contrast, platelet clusters were stable in size when metal ions were chelated, anti-ADAMTS13 IgG were added, or washed blood cells were perfused with purified VWF but no plasma. In the latter case, addition of recombinant ADAMTS13 reduced platelet cluster size by more than 70%. Incubating ADAMTS13 with VWF before perfusion did not prevent the initial platelet clustering, indicating that the enzyme may act on platelet-bound VWF under shear stress. At the concentrations tested, ADAMTS13 had no effect on platelet aggregates formed upon blood perfusion over collagen fibrils. ADAMTS13, therefore, may regulate thrombus size preferentially when the cohesion between platelets depends on VWF binding induced by pathologically elevated shear stress.     

Platelet factor 4 inhibits ADAMTS13 activity and regulates the multimeric distribution of von Willebrand factor

The efficiency of von Willebrand factor (VWF) in thrombus formation is related to its multimeric size, which is controlled by the protease ADAMTS13. However, it is not clear what regulates ADAMTS13 activity. In this study, we investigated whether PF4 could bind to VWF and inhibit ADAMTS13 activity. We found that PF4 binds to VWF and protects against ADAMTS13 activity. We also found that VWF-PF4 complexes circulate in patients with thrombotic thrombocytopenic purpura (TTP). Our data provides the first evidence that PF4 may have a novel role in regulating VWF multimers during primary haemostasis and thrombosis.     

The association between the L1565 variant of von Willebrand factor and susceptibility to proteolysis by ADAMTS13

The cysteine allele of the amino acid polymorphism (AAP) Y/C1584 in the A2 domain of von Willebrand factor (VWF) has been shown to correlate with enhanced VWF proteolysis by ADAMTS13. The frequencies and effect on VWF proteolysis of six reported AAP in VWF domains A1 and A2 were investigated. Only two AAP were variant: 4414 G/C (D/H1472) (allele frequency 0.86/0.14) and 4693 G/T (V/L1565) (allele frequency 0.92/0.08). D/H1472 had no apparent effect on VWF proteolysis. For V/L1565, a small but statistically significant increase in proteolysis was observed for V/L1565 VWF compared with V/V1565 VWF (p=0.0004).