vWF-cleaving Protease Activity Research Articles

Beneficial Effects of High-Density Lipoproteins on Acquired von Willebrand Syndrome in Aortic Valve Stenosis.

Infusions of apolipoprotein A-I (apoA-I), the major protein component of high-density lipoproteins (HDL), result in aortic valve stenosis (AVS) regression in experimental models. Severe AVS can be complicated by acquired von Willebrand syndrome, a haemorrhagic disorder associated with loss of high-molecular-weight von Willebrand factor (vWF) multimers (HMWM), the latter being a consequence of increased shear stress and enhanced vWF-cleaving protease (ADAMTS-13) activity. Although antithrombotic actions of HDL have been described, its effects on ADAMTS-13 and vWF in AVS are unknown. We assessed ADAMTS-13 activity in plasma derived from a rabbit model of AVS (n = 29) as well as in plasma collected from 64 patients with severe AVS (age 65.0 ± 10.4 years, 44 males) undergoing aortic valve replacement (AVR). In both human and rabbit AVS plasma, ADAMTS-13 activity was higher than that in controls (p < 0.05). Accordingly, AVS patients had less HMWM than controls (66.3 ± 27.2% vs. 97.2 ± 24.1%, p < 0.0001). Both ADAMTS-13 activity and HMWM correlated significantly with aortic transvalvular gradients, thereby showing opposing correlations (r = 0.3, p = 0.018 and r = -0.4, p = 0.003, respectively). Administration of an apoA-I mimetic peptide reduced ADAMTS-13 activity in AVS rabbits as compared with the placebo group (2.0 ± 0.5 RFU/sec vs. 3.8 ± 0.4 RFU/sec, p < 0.05). Similarly, a negative correlation was found between ADAMTS-13 activity and HDL cholesterol levels in patients with AVS (r = -0.3, p = 0.045). Our data indicate that HDL levels are associated with reduced ADAMTS-13 activity and increased HMWM. HDL-based therapies may reduce the haematologic abnormalities of the acquired von Willebrand syndrome in AVS.

Letter to the editor Cyclophosphamide-rescued plasmapheresis-unresponsive secondary thrombotic thrombocytopenic purpura caused by Sjögren’s syndrome

Letter to the editor Cyclophosphamide-rescued plasmapheresis-unresponsive secondary thrombotic thrombocytopenic purpura caused by Sjögren’s syndrome

Association of ADAMTS13 polymorphism with cerebral malaria

BackgroundCerebral malaria is one of the most severe manifestations of Plasmodium falciparum malaria. The sequestration of parasitized red blood cells (PRBCs) to brain microvascular endothelium has been shown to contribute to the pathophysiology of cerebral malaria. Recent studies reported increased levels of von Willebrand factor (VWF) and reduced activity of VWF-cleaving protease, ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), in patients with cerebral malaria.MethodsAssociation of six single nucleotide polymorphisms (SNPs) of the ADAMTS13 gene with cerebral malaria was examined in 708 Thai patients with P. falciparum malaria.ResultsAmong six SNPs, the derived allele of a SNP located in intron 28, rs4962153-A, was significantly associated with protection against cerebral malaria when 115 cerebral malaria patients were compared with 367 mild malaria patients (Fisher's exact P-value = 0.0057; OR = 0.27; 95% CI = 0.096-0.76). Significant association was also detected between 115 cerebral malaria and 593 non-cerebral malaria (226 non-cerebral severe malaria and 367 mild malaria) patients (Fisher's exact P-value = 0.012; OR = 0.30; 95% CI = 0.11-0.83).ConclusionsExcessive adhesion of PRBCs to the platelet-decorated ultra-large VWF (ULVWF) appears to enhance the sequestration of PRBCs to cerebral microvascular endothelium. The genetic association observed in the present study implies that the regulation of platelet-decorated ULVWF strings by ADAMTS13 may play a role in the development of cerebral malaria.

Screening for ADAMTS13 Gene Mutations in Thrombotic Thrombocytopenic Purpura Reveals a New Deletional Mutation.

Thrombotic Thrombocytopenic Purpura (TTP) is a rare severe thrombotic microangiopathy due to the presence of ultra large von Willebrand factor (UL-vWF) multimers in circulation. The aetiology has been linked to a congenital deficiency or inhibition of the vWF-cleaving protease (ADAMTS-13) activity by autoantibodies, which neutralise the enzymatic activity or bind the protease, increasing the plasmatic clearance (non-neutralising). The presence of autoantibodies can be associated with mutations and polymorphisms (SNPs) in the ADAMTS-13 gene. The goal of this study was to characterize the ADAMTS-13 in 4 female patients with acute TTP episodes. The clinical picture was characterized by neurological symptoms and microangiopathic hemolytic anemia (Hb 6 – 9 g/dL), thrombocytopenia (platelets 4 – 47 x109/L), elevated LDH (1874 – 4922 IU/mL). In one patient TTP episodes occurred during 4 pregnancies resulting in fetal deaths; one patient has systemic lupus. No precipitant factors were identified in the other 2 women; 1 of them had 3 TTP episodes.Methodology: ADAMTS-13 antigen and IgG anti-ADAMTS-13 were studied by ELISA (American Diagnostica). The vWF multimeric pattern was analyzed by SDS electroforesis, Western Blot, imunofixation and densitometry. Mutations screening along the 29 exons and boundaries of the ADAMTS-13 gene was performed by direct sequencing.Results: All the patients presented UL-vWF and autoantibodies anti-ADAMTS-13 and decreased ADAMTS-13 antigen. ADAMTS-13 gene studies identified SNPs in the four patients and mutations 764_776 del12 and R1096H, in heterozygosity, in the two patients with TTP associated with pregnancy and lupus, respectively.ADAMTS13ADAMTS13 genePatientsAgeTTPUL vWFAg (ng/ml)IgG (AU/ml)MutationModulators SNPsI73idiopatic+<62.518 (pos)No mutR7W (Htz); Q488E (Hm);II41Idiopatic*+12720.3 (pos)No mutR7W (Hm); Q488E (Hm) P618A (Hm); A732V (Hm)III32lupus+28430 (pos)E25, R1096H (Htz)R7W (Htz); Q488E (Hm)IV26Pregnancy**+<62.522.6 (pos)E7, 764_776del12 (Htz)R625H (Hm)Reference range: Ag: 485–1242 ng/ul; IgG: cutoff >13.9 AU/ml; *3 episodes; **4 episodesDiscussion: PTT in these four patients was associated with antibodies anti-ADAMTS-13 and low levels of protein. Identification of the R7W and Q488E SNPs, described as positive modulators of protein secretion, on Patient I suggests the presence of the non-neutralising type of antibodies responsible for an increased plasmatic protein clearance. The positive modulation of the same SNPs on Patient II and III may be abolished by the presence of SNPs P618A and A732V (Camilleri et al; Pleimaueur et al) on Patient II and the mutation R1096H (Meyer et al) on Patient III. The not previously described mutation 764_776del12, identified on Patient IV, is likely to disturb the protein aminoacid composition, compromising the protease structure and function. This patient had a PTT episode in each of her four pregnancies.

Thrombotic thrombocytopenic purpura during immune reconstitution

The emergence of newer antiretroviral agents has revolutionized the treatment of patients struggling with multidrug-resistant strains of HIV. As a result, we are witnessing a higher incidence of immune reconstitution inflammatory syndrome, most commonly associated with mycobacterial, cryptococcal, herpetic and other infections, in addition to autoimmune conditions such as photodermatitis, systemic lupus erythematosus [1], Grave's thyroiditis [2] and sarcoid [3]. The pathogenesis of immune reconstitution inflammatory syndrome is unclear, but has been proposed to be a combination of thymic-independent homeostatic peripheral expansion, immune dysregulation between effector and regulatory T cells, increased inflammatory cytokines and dysregulated dendritic cells [4,5]. We present the case of a 48-year-old African-American man, with multiple drug-resistant HIV-1 infection, who was admitted to the hospital with acquired thrombotic thrombocytopenic purpura (TTP) 4 weeks after darunavir/norvir and enfuvirtide was added to tenofovir/emtricitabine. The patient had a past medical history only significant for chronic hepatitis B, with an undetectable viral load. After 4 weeks on the new regimen, his CD4 cell count increased from 94 (10%) to 351 (16.7%) cells/μl and his viral load decreased from more than 100 000 to 151 copies/ml. He presented with a non-productive cough, frontal headache, dark urine, dyspnea on exertion, myalgias and arthralgias from a previously asymptomatic state. Laboratory work-up (baseline in parenthesis) showed a hemoglobin level of 7.4 g/dl (11.2), platelets of 22 × 103/μl (221), lactate dehydrogenase 2450, creatinine 1.8 mg/dl (1.2), and a peripheral smear showed many schistocytes. An assay (Esoterix Inc., Austin, Texas, USA) showed the von Willebrand factor (vWF) cleaving protease to be deficient (< 8%), and the presence of a disintegrin-like and metalloprotease with thrombospondin-type 1 motif-13 (ADAMTS-13) vWF cleaving protease inhibitor (1.2 inhibitor units). No other inciting event was identified after extensive work-up. All his antiretroviral drugs were discontinued except for tenofovir/emtricitabine, whereas plasmapharesis, vincristine and prednisone were initiated. He had a full and uneventful recovery one month later. Subsequently, our patient was successfully restarted on antiretroviral therapy (darunavir/norvir and tenofovir/emtricitabine) without a recurrence of TTP, while experiencing increasing CD4 cell counts and decreasing viral loads. Although TTP has been described in relation to HIV [6], to our knowledge this is the first reported case of acquired TTP in the setting of immune reconstitution. Our patient had a low absolute CD4 cell count and CD4 cell percentage at baseline, followed by a ‘fulminant’ CD4 cell count recovery and viral suppression shortly after starting his new regimen. A Medline search failed to identify any association between TTP and antiretroviral agents, i.e. T-20, darunavir and others. Although a causal mechanism cannot be established from one case, we propose two insults accounting for a major alteration of vWF homeostasis based on previous descriptions of TTP [7]. The first alteration was caused by inflammatory cytokines (IL-6, IL-8, tumour necrosis factor alpha and others) that stimulate the ultralarge, hyperreactive vWF release from endothelial cells. The second insult is immune deregulation mediated by IL-6 and other autoantibodies that inhibit (ADAMTS-13) vWF cleaving protease activity, abrogating the ability of ADAMTS-13 to cleave ultralarge vWF, leading to the accumulation of ultralarge vWF in plasma and on the surface of endothelial cells. Could these two hits combined account for the high tendency for platelet aggregation and thrombosis that patients might be at risk of during the acute phase of immune reconstitution? The second reason we believe this case to be notable is its possible implication in the increase in cardiovascular events after immune reconstitution (unpublished). We believe that further studies should investigate the role of thrombosis in the increased cardiovascular events after acute immune reconstitution.

Altered ADAMTS-13 Expression in Human Umbilical Vein Endothelial Cells.

von Willebrand factor (vWF), as secreted by endothelial cells (ECs), is a highly thrombogenic, ultra-large multimeric protein which promotes platelet adhesion and clot formation. This ultra-large vWF would lead to platelet aggregation even without vascular injury if not for the activity of a plasma vWF-cleaving protease, ADAMTS-13, which cleaves the ultra-large vWF multimers upon secretion into the more moderately-sized, less thrombogenic multimers commonly found circulating in normal plasma. The production of ADAMTS-13 was first described in hepatic stellate cells, but has since been found to be produced in a variety of tissues and cell types including ECs. Production of ADAMTS-13 by ECs is of interest as the newly secreted ultra-large vWF is immediately cleaved on the surface of ECs by ADAMTS-13. Quiescent ECs normally exist as a confluent non-thrombogenic monolayer lining the lumen of the vasculature. Damage to the EC monolayer results in cell activation, stimulating not only thrombosis but proliferation as the ECs attempt to prevent blood loss and repair vascular wall damage. To assess the production of ADAMTS-13 by human umbilical vein ECs, as measured by a commercially-available ELISA (American Diagnostica Inc., Stamford, CT), cellular supernatants were collected and cellular proteins extracted from either confluent ECs (EC monolayers), damaged confluent ECs (EC monolayers which had narrow strips of cells removed with a sterile pipette tip), or subconfluent ECs (50% confluence). After four hours, supernatants from subconfluent ECs contained 2.5X as much ADAMTS-13 (161.5±29.1 pg/ml/μg cellular protein; p<0.002) as compared with either confluent or damaged confluent ECs (60.2±22.5 and 56.0±19.2 pg/ml/μg cellular protein, respectively). Similarly, cell lysates from subconfluent ECs contained ∼2.5X as much ADAMTS-13 (645.1±73.1 pg/ml/μg cellular protein; p<0.002) as compared with either confluent or damaged confluent ECs (257.8±32.8 and 311.3±29.3 pg/ml/μg cellular protein, respectively). In time-course experiments, confluent, damaged confluent, and subconfluent ECs were fixed 4, 24, 48, 72, or 96 hours following injury (producing the damaged confluent ECs). ADAMTS-13 expression was subsequently detected by immunohistochemistry using a rabbit polyclonal anti-human ADAMTS-13 antibody directed against the metalloproteinase domain (Abcam Inc., Cambridge, MA) and a fluorescent secondary antibody. Subconfluent ECs expressed more intense staining for ADAMTS-13 throughout the time course when compared with the confluent ECs. By 72 hours, the damaged confluent ECs began expressing comparable intense staining, particularly in regions where the ECs were reestablishing the monolayer. Previous work has suggested that the hepatic stellate cells, as well as the other cell types which express ADAMTS-13, produce this protease constitutively and that this production is not otherwise regulated. The data presented herein demonstrate that ADAMTS-13 production, at least in ECs, is affected by the growth state of the ECs. This enhanced production of ADAMTS-13 may limit the extent of platelet thrombosis at sites of vascular injury.

Alterations of ADAMTS-13 Activity as a Common Indicator of the Endothelial Dysfunction Developing in Different Thrombotic Microangiopathies.

Injury to the endothelial cell is a key factor for the initiation and development of thrombotic microangiopathies (TMA). Endothelial dysfunction implies alterations of the von Willebrand factor (vWF) pathway. Severe deficiency of the vWF-cleaving protease activity has been traditionally associated with thrombotic thrombocytopenic purpura (TTP). However, there is ongoing evidence indicating that ADAMTS-13 may be a causal factor in the pathogenesis of other TMA.We evaluated whether ADAMTS-13 deficiency and autoantibodies inactivating the protease were prevalent in TMA with different physiopathological origin. Patients included in the study presented TMA associated with: TTP (n=13), hemolytic uremic syndrome (HUS) (n=4), gastrointestinal angiodysplasia (GA) (n=11), or stem cell transplantation (SCT) (n=3). We measured ADAMTS-13 activity and antigen (ADAMTS-13:Ag), the presence and inhibitory activity of autoantibodies to the protease, vWF:Ag, and vWF:multimeric pattern. ADAMTS-13 activity and inhibitory activity of autoantibodies were analyzed using a modification of the immunoblotting assay described by Furlan et al (1996). ADAMTS-13:Ag and the presence of IgG were measured by ELISA kits.The vWF:multimeric pattern was resolved by electrophoresis in agarose gels.In patients with TTP, undetectable ADAMTS-13 activity was found in 10 out of 13 patients, 9 of which had IgG against the metalloprotease and reduced ADAMTS-13:Ag levels. Plasma exchange normalized the ADAMTS-13:Ag levels and decreased IgG title. Interestingly, one case of TTP with autoantibodies was associated with clopidogrel treatment. Levels of vWF:Ag in these patients varied from 50 to 180%.As for patients with HUS, in 2 out of 4 ADAMTS-13 activity was absent and ADAMTS-13:Ag was reduced, but no IgG to the metalloprotease was detected. It should be pointed out that one of the cases of HUS was subsequent to cocaine abuse. In TMA associated with SCT and GA, ADAMTS-13 activity was around 20% in all the patients studied, although levels of the metalloprotease were within the control range (700–1400ng/ml). vWF:Ag levels were found to be significantly increased (to around 200%) in the former group of patients.In summary, presence of autoantibodies to ADAMTS-13 concurred with decreased antigen levels, reinforcing the hypothesis of an accelerated clearance of the metalloprotease in vivo. Among the patients studied, we found a clopidogrel associated TTP and a cocaine caused HUS. Presence of antibodies to ADAMTS-13 associated with clopidogrel treatment would suggest an immunological mechanism, which cannot be ruled out in the case of cocaine associated HUS. Overall, our results indicate that the absence of ADAMTS-13 activity is not specific for TTP. A reduced activity of the metalloprotease may be an indicator of endothelial dysfuntion as a common feature in different TMA.

Fatal relapse of thrombotic thrombocytopaenic purpura after cardiac surgery in patient with congenital absence of vWF-cleaving protease activity

Fatal relapse of thrombotic thrombocytopaenic purpura after cardiac surgery in patient with congenital absence of vWF-cleaving protease activity

Analysis on the Molecular Species and Concentration of Circulating ADAMTS13 in Blood

ADAMTS13 is the metalloprotease responsible for the proteolytic degradation of von Willebrand factor (VWF). A severe deficiency of this VWF-cleaving protease activity causes thrombotic thrombocytopenic purpura. This protease, comprising 1,427 amino acid residues, is composed of multiple domains, i.e., a preproregion, a metalloprotease domain, a disintegrin-like domain, a thrombospondin type-1 motif (Tsp1), a cysteine-rich domain, a spacer domain, seven Tsp1 repeats, and two CUB domains. We prepared one polyclonal and seven monoclonal antibodies recognizing distinct epitopes spanning the entire ADAMTS13 molecule. Of these antibodies, two of the monoclonal ones, which recognize the disintegrin-like and cysteine-rich/spacer domains, respectively, abolished the hydrolytic activity of ADAMTS13 toward both a synthetic substrate, FRETS-VWF73, and the natural substrate, VWF. In addition, these antibodies blocked the binding of ADAMTS13 to VWF. These results revealed that the region between the disintegrin-like and cysteine-rich/spacer domains interacts with VWF. Employing these established polyclonal and monoclonal antibodies, we examined the molecular species of ADAMTS13 circulating in the blood by immunoprecipitation followed by Western blot analysis, and estimated the plasma concentration of ADAMTS13 by enzyme-linked immunosorbent assay. These studies indicated that the major fraction of ADAMTS13 in blood plasma consisted of the full-length form. The concentration of ADAMTS13 in normal plasma was approximately 0.5-1 microg/ml.

Shigatoxin Triggers Thrombotic Thrombocytopenic Purpura in Genetically Susceptible ADAMTS13-Deficient Mice.

The life threatening disease TTP is associated with ultra-large von Willebrand Factor multimers (UL-VWF) in the circulation due to inherited or acquired deficiency of the ADAMTS13 metalloprotease. Here we show that ADAMTS13-deficient mice generated by gene targeting are viable and exhibit normal survival through 2 years of age. Despite the absence of VWF-cleaving protease activity (<1% of normal), wild-type and ADAMTS13-deficient plasma exhibit identical VWF multimer distributions, and Adamts13−/− mice develop spontaneous TTP at an extremely low rate (2 cases out of 358 mice). However, intravital microscopy demonstrated that VWF-mediated platelet-endothelial interactions are significantly prolonged in Adamts13−/− mice. These observations suggested that additional environmental triggers and/or genetic modifying factors may be required to bring about TTP in the setting of ADAMTS13 deficiency. To address the effect of VWF level on development of TTP, Adamts13−/− mice were crossed to mice of the CASA/Rk strain which exhibit markedly elevated plasma VWF levels. Resulting CASA/Adamts13−/− mice demonstrated plasma VWF ranging from 150% to 600% of C57BL/6 controls, and we found that 21% of these mice were thrombocytopenic at baseline (vs. 0% of controls). Introduction of the CASA/Rk genetic background also resulted in the appearance of UL-VWF in CASA/Adamts13−/− mice, further prolonged VWF-mediated platelet-endothelial cell interactions, increased the rate of spontaneous TTP, and markedly decreased survival. Challenge of CASA/Adamts13−/− mice with shigatoxin (derived from bacterial pathogens associated with the related human disease hemolytic uremic syndrome) resulted in a striking syndrome closely resembling human TTP, with thrombocytopenia, profound microangiopathic hemolytic anemia, and platelet- and VWF-thrombi seen in multiple organs. Surprisingly, we observed no correlation between plasma VWF level and severity of TTP, implying the existence of TTP-modifying genes distinct from VWF. Our laboratory is pursuing the identification of these genes which may provide insight into the pathogenesis and treatment of TTP in humans. Finally, our data also suggest that microbial-derived toxins, or possibly other sources of endothelial injury, may be among the key factors required to trigger acute TTP in the setting of ADAMTS13 deficiency.

Platelets Treated with Ticlopidine Are Less Reactive to Unusually Large von Willebrand Factor Multimers than Are Those Treated with Aspirin under High Shear Stress

Much attention has recently been focused on the interaction between unusually large von Willebrand factor multimers (UL-VWFM) and platelets under high shear stress in pathological thrombus formation. The antiplatelet drugs acetylsalicylic acid (aspirin) and a thienopyridine derivative (ticlopidine) are commonly used to treat cerebral ischemia but exert different effects on high-shear-stress-induced platelet aggregation (H-SIPA) in the plasma. To examine the effects of these drugs in the absence of plasma factors, we studied H-SIPA using washed platelets (WPs) and purified UL-VWFM. WPs were prepared from the blood of 9 aspirin-treated and 11 ticlopidine-treated patients with cerebral ischemia, and H-SIPA in the presence of UL-VWFM was measured using a cone plate aggregometer. Plasma levels of VWF antigen with its multimer analysis, ristocetin cofactor and VWF-cleaving protease (ADAMTS13) activity were also measured. Forty-six healthy volunteers from 2 age groups, 20–40 years (n = 20) and 41–60 years old (n = 26), were also tested as controls. H-SIPA was significantly inhibited for ticlopidine-treated platelets, but it was observed to a lesser extent for aspirin-treated platelets. For both groups, no difference in the plasma levels of VWF antigen, ristocetin cofactor and ADAMTS13 activity was noted. All patients possessed UL-VWFM, and it was detected in healthy volunteers with increasing frequency with increasing age. Under plasma-free conditions, platelets from aspirin-treated patients exhibit marginal but significant inhibition of H-SIPA. Furthermore, the presence of UL-VWFM in the plasma of patients and normal volunteers is directly related to their age rather than being a consequence of underlying disease.

Coagulation factor levels in cryosupernatant prepared from plasma treated with amotosalen hydrochloride (S‐59) and ultraviolet A light

The standard treatment for thrombotic thrombocytopenic purpura (TTP) is plasma exchange with fresh-frozen plasma (FFP). Exposure to large volumes of FFP increases the risk of transfusion-transmitted infections. Cryosupernatant (CSP) offers a theoretical advantage over FFP, because it lacks the large von Willebrand factor (VWF) forms implicated in the pathogenesis of TTP. This study compared the hemostatic variables of CSP prepared from FFP treated with a photochemical pathogen inactivation process to CSP prepared from conventional FFP. Forty CSP units were prepared from North American blood group A donor FFP. Twenty-one of the FFP units were individually treated with amotosalen hydrochloride (S-59) and ultraviolet A light (test, photochemically treated FFP), and 19 units were not treated (control, FFP). Hemostatic variables of test and control CSP were similar and within reported ranges for conventional FFP with the exception of those properties depleted in CSP. VWF-cleaving protease activity (VWF:CP) and protein S (PS) levels (total and free antigen and activity) were within the conventional FFP reference range for test and control CSP. There were statistical differences between test and control CSP for alpha(2)-antiplasmin, antithrombin, protein C, and VWF:CP on a per-volume basis, but all levels were within the reference range for FFP, and the differences were not significant when expressed per gram of CSP protein. S-59-treated CSP retained adequate levels of critical plasma proteins for plasma exchange therapy in acute TTP. The data indicate good preservation of hemostasis control proteins such as PS, alpha(2)-antiplasmin, and VWF:CP activity (ADAMTS13).

The quality of fresh‐frozen plasma produced from whole blood stored at 4°C overnight

The aim of this study was to assess whether the quality of FFP produced from whole blood stored at 4 degrees C overnight is adequate for its intended purpose. Fresh-frozen plasma (FFP) separated from whole blood (n = 60) leukodepleted (LD) after storage at 4 degrees C overnight (18-24 hr from donation, Day 1 FFP) was compared with that LD within 8 hours of donation (Day 0 FFP, the current standard method). In more than 95 percent of Day 1 FFP units, levels of factor (F) II, FV, FVII, FVIII, F IX, FX, FXI, and FXII were greater than 0.50 U per mL except for von Willebrand factor (VWF) antigen and FVIII, where 92 and 87 percent of units, respectively, contained greater than 0.50 IU per mL. Compared with historical data on FFP stored for 8 hours, fibrinogen, FV, FVIII, and FXI were reduced by 12, 15, 23, and 7 percent, respectively, but other factors were not significantly reduced. Levels of VWF-cleaving protease activity were not different between FFP prepared from paired units of blood (n = 3) held for 8 or 24 hours, but were below the reference range in an additional 2 of 6 units held for 24 hours. The activities of protein S, protein C, antithrombin III, and alpha(2)-antiplasmin were reduced by less than 10 percent in Day 1 FFP (n = 20), but with final levels above the lower limit of the normal range in greater than 95 percent of units. Activated FXII antigen was not significantly raised in plasma stored for 18 to 24 hours, but levels of prothrombin fragment 1 + 2 were slightly increased (0.88 ng/mL, 18-24 hr; 0.65 ng/mL, < 8 hr). These data suggest that there is good retention of relevant coagulation factor activity in plasma produced from whole blood stored at 4 degrees C for 18 to 24 hours and that this would be an acceptable product for most patients requiring FFP.

Role of Chloride Ions in Modulation of the Interaction between von Willebrand Factor and ADAMTS-13

The degradation of von Willebrand factor (VWF) depends on the activity of a zinc protease (referred to as ADAMTS-13), which cleaves VWF at the Tyr(1605)-Met(1606) peptide bond. Little information is available on the physiological mechanisms involved in regulation of AD-AMTS-13 activity. In this study, the role of ions on the ADAMTS-13/VWF interaction was investigated. In the presence of 1.5 m urea, the protease cleaved multimeric VWF in the absence of NaCl at pH 8.00 and 37 degrees C, with an apparent k(cat)/K(m) congruent with 3.4 x 10(4) M(-1) s(-1), but this value decreased by approximately 10-fold in the presence of 0.15 M NaCl. Using several monovalent salts, the inhibitory effect was attributed mostly to anions, whose potency was inversely related to the corresponding Jones-Dole viscosity B coefficients (ClO(4)(-) > Cl(-) > F(-)). The specific inhibitory effect of anions was due to their binding to VWF, which caused a conformational change responsible for quenching the intrinsic fluorescence of the protein and reducing tyrosine exposition to bulk solvent. Ristocetin binding to VWF could reduce the apparent affinity and reverse the inhibitory effect of chloride. We hypothesize that, after secretion into the extracellular compartment, VWF is bound by chloride ions abundantly present in this milieu, becoming unavailable to proteolysis by AD-AMTS-13. Shear forces, which facilitate GpIbalpha binding (this effect being artificially obtained by ristocetin), can reverse the inhibitory effect of chloride, whose concentration gradient across the cell membrane may represent a simple but efficient strategy to regulate the enzymatic activity of ADAMTS-13.

Dissociation between the level of von Willebrand factor‐cleaving protease activity and disease in a patient with congenital thrombotic thrombocytopenic purpura

Decreased von Willebrand factor (VWF)-cleaving protease activity (<5%) has been implicated in patients with congenital thrombotic thrombocytopenic purpura-hemolytic uremic syndrome (Upshaw-Schulman syndrome) and associated with mutations within the ADAMTS13 gene. In this report, we describe longitudinal studies in a patient with congenital TTP who ultimately developed end-stage renal failure and required plasma therapy from infancy. The patient was deficient in plasma high molecular weight (HMW)-VWF multimers during acute disease but had increased amounts of the HMW-VWF multimers during periods of remission. DNA analysis of this patient detected homozygosity for the R692C mutation on exon 17 of the ADAMTS13 gene, previously linked to congenital TTP. The level of VWF-cleaving protease activity in the patient was remarkably low (<5%) throughout her disease, even after she entered complete remission. However, despite no improvement in the level of VWF-cleaving protease activity, this patient had complete resolution of disease following splenectomy and commencing hemodialysis, without need for ongoing plasma therapy. The patient has remained in remission for over 4 years. These observations suggest that there are other factors in conjunction with severe deficiency of VWF protease activity that participate in the platelet-mediated thrombotic complications and other disease manifestations of congenital TTP. In addition, it is possible that splenectomy could be an effective treatment option for some patients with severe, congenital TTP.

Additional Environmental and/or Genetic Factors Are Required to Trigger TTP in ADAMTS13-Deficient Mice.

Thrombotic Thrombocytopenic Purpura (TTP) is a life threatening systemic illness characterized by the formation of platelet-rich thrombi in the microcirculation and the clinical pentad of fever, hemolytic anemia, thrombocytopenia, neurological symptoms, and renal dysfunction. TTP is associated with ultra-large von Willebrand Factor multimers (UL-VWF) in the circulation due to deficiency of the ADAMTS13 metalloprotease. ADAMTS13 gene mutations account for most if not all cases of familial TTP, and autoantibodies to ADAMTS13 underlie most cases of acquired TTP. To further explore the pathogenesis of TTP in vivo, ADAMTS13 deficient mice were generated by gene targeting to remove exons 1–6 which encode most of the protease domain of ADAMTS13. Although the resulting homozygous null mice (Adamts13−/−) have lost all specific VWF-cleaving protease activity (< 1% of control), embryonic development is normal, and the null mice are born at the expected Mendelian frequency. Analysis of baseline hematologic parameters and peripheral blood smears revealed no difference between Adamts13−/− mice and wild-type littermates, with no evidence for thrombocytopenia or microangiopathic hemolytic anemia. Pathologic survey of multiple tissues revealed only normal histology and no evidence for platelet or VWF-rich thrombi in the vasculature. Despite the absence of VWF-cleaving protease activity, plasma from wild-type and ADAMTS13-deficient mice exhibited identical VWF multimer size distributions. However, VWF multimers from both wild-type and Adamts13−/− mice were observed to be considerably larger than those from normal human plasma, and equivalent in size to UL-VWF seen in plasma from familial TTP patients. Challenge of Adamts13−/− mice by injection with endotoxin, and genetic crosses to mice with markedly elevated VWF levels (CASA/Rk), failed to induce findings consistent with TTP. However, treatment with verotoxin-2 (a bacterial endothelial toxin important in the pathogenesis of the hemolytic uremic syndrome), caused thrombocytopenia in 6 of 11 Adamts13−/− mice (vs. 3 of 11 wild-type controls, p < 0.07) and mortality at 6 days in 9 of 11 Adamts13−/− mice (vs. 6 of 11 controls, p < 0.02). Examination of peripheral blood from one of the Adamts13−/− mice at 8 days following verotoxin administration demonstrated marked microangiopathic changes. In conclusion, mice with targeted disruption of the Adamts13 gene do not develop TTP spontaneously, suggesting the requirement for additional environmental triggers or genetic modifiers. Though some humans with congenital ADAMTS13 deficiency have been reported to remain asymptomatic for many years, the increased size of the baseline VWF multimer distribution in mice may indicate a higher threshold for VWF-ADAMTS13 interaction which may be protective for TTP. Our results also suggest that microbial-derived toxins, or other sources of endothelial injury, may be one of the key environmental triggers responsible for the lack of spontaneous TTP findings in the ADAMTS13-deficient mice, and possibly for the intermittent symptoms seen in humans.

ADAMTS13 Activity in Sickle Cell Disease: A Role in the Development of Clinical Vaso-Occlusion?.

Endothelial cell activation occurs continuously in sickle cell disease (SCD). As VWF and especially the ultra large VWF multimers, released from activated endothelial cells, can serve as bridging proteins between the vessel wall and sickle red blood cells, we determined ADAMTS13 (the vWF-cleaving protease) activity as well as VWF:Rcof and VWF:Ag levels in SCD. Fourteen sickle cell patients (13 HbSS, 1 HbSC; 39 samples collected) and 9 HbAA controls were included. VWF:Rcof and VWF:Ag were assessed by commercially available assays, ADAMTS13 activity was determined by quantitative immunoblotting of proteolysed VWF-substrate. VWF:Rcof and VWF:Ag levels were higher in patients, as compared to controls. ADAMTS13 activity ranged from 45–128% in asymptomatic patients, from 38–205 in patients during acute vaso-occlusive events (VOE) and from 43-75% in controls. The ratios of ADAMTS13 to vWF:Ag levels and activity were lower in patients (SCD asymptomatic: 0.44 [interquartile range 0.34–0.55], SCD acute VOE: 0.29 [0.21–0.43], HbAA: median 0.61 [0.33–1.23], Kruskar Wallis test p=0.003). The ADAMTS13 - VWF:Rcof and the ADATS13 - VWF:Ag ratios dropped during acute vaso-occlusive events in 3 of 6 patients. In patients from whom multiple samples were collected (n=4), ADAMTS13 activity and VWF:Ag levels (and to a lesser extent VWF:Rcof) were inversely related. One patient showed no change in ADAMTS13 activity despite strong increments in VWF:Ag and VWF:Rcof during vaso-occlusive pain crises prior to his death (pulmonary embolism). VWF:Ag levels at initial presentation of acute VOE were related to the duration of pain (rs=0.53, p=0.02), whereas the ratios of ADAMTS13 to VWF:Ag levels and ADAMTS13 to VWF:Rcof were inversely related hereto (rs=−0.67, p=0.002, rs=−0.53, p=0.02 respectively). These data demonstrate that ADAMTS13 activity is related to VWF:Ag levels and VWF:Rcof in SCD and that the role of ADAMTS13 and VWF in the pathophysiology of SCD deserves further evaluation.

High Shear Stress and Immobilization of Von Willebrand Factor Promotes the VWF-Cleaving Protease Activity as Assessed by the Cone and Plate(let) Analyzer.

The metalloprotease ADAMTS-13 cleaves von Willebrand factor (VWF) that is released from endothelial cells as a large multimeric protein. However, the specific physiological conditions required for the function of this von Willebrand factor-cleaving protease (VWF-CP) are not yet established. In this study we determined the optimal conditions for the VWF-CP activity using the Cone and Plate(let) Analyzer (CPA). Proteolysis of a soluble recombinant VWF by a recombinant VWF-CP in the absence or the presence of BaCl2 (for induction of VWF-CP) was performed under static and flow (2050 s−1) conditions. The resulted fragments of the soluble VWF were immobilized on a polystyrene surface and non-adhering sites were blocked with 1% BSA. In parallel, polystyrene surface immobilized VWF was similarly treated by VWF-CP. The immobilized cleaved VWF fragments by the above protocols served as a substrate for citrated whole blood platelet adhesion under flow (2050 s−1). Reduction of platelet adhesion (surface coverage, SC) in BaCl2 treated compared to untrerated sample reflects the degree of VWF-CP activity.Platelet adhesion (SC, %) to VWF proteolytic products generated under the indicated conditions:Static w/o BaCl2Static + BaCl2Flow w/o BaCl2Flow + BaCl2Soluble VWF15.0 ± 3.411.1 ± 2.713.6 ± 2.28.0 ± 2.1Immobil. VWF19.1 ± 3.012.0 ± 4.16.5 ± 1.11.0 ± 0.6Maximal VWF-CP activity as reflected by maximal reduction (85%) of platelet adhesion was observed when immobilized VWF was treated by VWF-CP under flow. Minimal effect of VWF-CP activity was observed when soluble VWF was treated under static condition (26% reduction). Intermediate effect was observed with soluble VWF under flow (41%) and with immobilized VWF under static condition (37%). It should be noted that in the case of immobilized VWF application of flow alone (without BaCl2) was sufficient to induce a remarkable decrease of platelet adhesion (66%). In conclusion, both immobilization of VWF and high shear stress are important conditions for VWF-CP function, suggesting that stretching of immobilized VWF under flow exposes the VWF-cleavage site and thereby may serve as a control mechanism of platelet adhesion at the site of thrombus formation.

Simplified assay for VWF cleaving protease (ADAMTS13) activity and inhibitor in plasma.

The absence of VWF cleaving protease activity (VWFcp, ADAMTS13) has recently been identified as a central component in the pathogenesis of TTP. Several assays for the measurement of ADAMTS13 activity have been described, but most are cumbersome and employ techniques not easily adapted to routine laboratories. Thus, ADAMTS13 assays are available at only a few reference or research laboratories. Prompt identification of absent or impaired ADAMTS13 activity could prove invaluable to clinical decision-making regarding diagnosis and treatment of suspected TTP patients. We describe an assay for ADAMTS13 that uses a commercial source of VWF substrate and obviates dialysis for sample and substrate preparation. Patient and normal plasma are prepared by desalting over Micro-Spin columns, and barium is added to activate the cleaving protease. Humate-P(R) is prepared over a desalting column and reconstituted in urea prior to use as the exogenous VWF substrate. Desalted plasma is mixed with prepared substrate, and digestion is carried out at 37 degrees C. Maximum sensitivity to very low levels of enzyme activity is achieved by using undiluted plasma and allowing digestion to proceed for 14 hr. The extent of VWF multimer cleavage is then demonstrated by Western blot. We used this assay to analyze VWF cleaving protease activity in plasma samples from 30 patients treated at our institution for TTP. Plasma from patients lacking ADAMTS13 was incubated with PNP and then analyzed by the same methods to determine the presence of an inhibitor. Our results indicate that the assay is suitable for providing timely information regarding ADAMTS13 activity for the diagnosis and treatment of patients with suspected TTP.

Cloning, expression, and functional characterization of the von Willebrand factor–cleaving protease (ADAMTS13)

Deficient von Willebrand factor (VWF) degradation has been associated with thrombotic thrombocytopenic purpura (TTP). In hereditary TTP, the specific VWF-cleaving protease (VWF-cp) is absent or functionally defective, whereas in the nonfamilial, acquired form of TTP, an autoantibody inhibiting VWF-cp activity is found transiently in most patients. The gene encoding for VWF-cp has recently been identified as a member of the metalloprotease family and designated ADAMTS13, but the functional activity of the ADAMTS13 gene product has not been verified. To establish the functional activity of recombinant VWF-cp, we cloned the complete cDNA sequence in a eukaryotic expression vector and transiently expressed the encoded recombinant ADAMTS13 in HEK 293 cells. The expressed protein degraded VWF multimers and proteolytically cleaved VWF to the same fragments as those generated by plasma VWF-cp. Furthermore, recombinant ADAMTS13-mediated degradation of VWF multimers was entirely inhibited in the presence of plasma from a patient with acquired TTP. These data show that ADAMTS13 is responsible for the physiologic proteolytic degradation of VWF multimers.