Ultra-large Von Willebrand Factor Multimers Research Articles

N-Acetyl Cysteine Reduces von Willebrand Factor Multimer Size In Vitro and In Vivo.

von Willebrand factor (VWF) shares a similar domain structure with many polymeric mucins, including the presence of D domains and a C-terminal cysteine knot, which allows these molecules to form polymeric structures that can reach immense sizes. Precipitation of polymeric lung mucins complicates the clinical course of cystic fibrosis and chronic obstructive lung disease, and in both cases the viscosity of the inspissated mucus can be reduced by treatment with N-acetyl cysteine (NAC), which presumably reduces the size of the mucin polymers by reducing sensitive disulfide bonds. Because of the similarity of VWF and mucin multimers, we examined whether NAC could also reduce VWF multimer size both in vitro and in vivo. In vitro, we incubated NAC at different concentrations and for different times with ultra-large VWF multimers (ULVWF) isolated from endothelial cell supernatant and examined the effect on multimer size using agarose gel electrophoresis. NAC reduced ULVWF size in a time- and concentration-dependent manner, with the peak effect reached at 5 min and at concentration of 0.5 mM. We then examined the effect of NAC on ULVWF/platelet “strings” formed on the surface of histamine-activated endothelial cells by perfusing the strings with NAC solutions. At 1 mM, NAC eliminated almost all of the adherent strings within 5 minutes. We next examined the effect of NAC in vivo by following VWF multimer size with time in C57B/6 injected with NAC either intraperitoneally or intravenously. NAC, at a single dose of 500 mg/kg, induced a sustained reduction in VWF multimer size in the treated mice within 4 hours after injection. The effects lasted up to 8 hours. These results suggest that NAC may be a rapid, safe, and effective treatment for patients suspected of suffering from thrombotic thrombocytopenic purpura, a disorder characterized by a failure to process ULVWF.

A Threshold Level of Von Willebrand Factor Is Required for Disease Pathogenesis in a Mouse Model of TTP.

Thrombotic Thrombocytopenic Purpura (TTP) is a life threatening systemic illness associated with inherited or acquired deficiency of the ADAMTS13 metalloprotease. ADAMTS13 deficiency results in the appearance of ultra-large multimers of von Willebrand Factor (UL-VWF) in the circulation, which are thought to play a central role in the formation of the platelet-rich thrombi which are the pathological hallmark of this disease. Despite the critical role postulated for VWF in TTP pathogenesis, direct experimental evidence in support of this hypothesis is lacking. Recently we demonstrated that a TTP-like syndrome can be induced in ADAMTS13-deficient mice of a mixed CASA/Rk genetic background (Adamts13CASA−/−) by the bacterial agent shigatoxin. As pure CASA/Rk mice exhibit VWF levels 5 to 10-fold higher than most of the common strains of laboratory mice, we hypothesized that increased plasma VWF levels in the Adamts13CASA−/− mice (which ranged from 200–600% of C57BL/6) were responsible for their increased TTP susceptibility. Surprisingly however, plasma VWF levels did not correlate with the degree of thrombocytopenia or hemolytic anemia induced in Adamts13CASA−/− mice by shigatoxin challenge, with mice inheriting the lowest VWF levels having TTP pathology equal to those inheriting the highest levels. To further examine the requirement for VWF in TTP pathogenesis, we generated ADAMTS13-deficient mice on a susceptible CASA/Rk genetic background that were also either haploinsufficient (Vwf+/−) or completely deficient (Vwf−/−) in VWF. Absolute deficiency of VWF resulted in complete protection from shigatoxin-induced thrombocytopenia. However, thrombocytopenia was consistently observed in Vwf+/− mice similar to that previously reported in Adamts13CASA−/− mice with wild-type VWF. Importantly, again no correlation was observed between VWF level and degree of thrombocytopenia among the ADAMTS13-deficient mice that were Vwf haploinsufficient (Vwf+/−), confirming our previous observation. In summary, we now have demonstrated an absolute requirement for VWF in the pathogenesis of TTP in this mouse model system. Together with the observations that increasing VWF levels do not correlate with increasing TTP pathology, these results suggest that a threshold level of VWF is required for induction of TTP, but that further increases of VWF level do not result in worsening disease. Though these findings remain to be confirmed in humans, our data suggest that the wide variation in plasma VWF levels in the human population will not be a significant determinant of TTP susceptibility or disease severity.

Von Willebrand Factor in Sickle Cell Disease - Relationship to Sleep Hypoxia.

Introduction:Despite the intriguing in vitro data implicating vWF in sickle-RBC-endothelial adhesion and in animal models of vasoocclusion, there are few clinical studies characterizing the role of vWF in SCD pathophysiology.Rationale: Plasma vWF is mainly endothelial-derived, upto 95% of newly synthesized vWF being secreted constitutively. Since in vitro studies have shown that hypoxia mediates release of ultra-large vWF multimers stored in endothelial Weibel Palade bodies, we hypothesized that changes in the plasma vWF profile of patients with sickle cell disease (SCD) could potentially be related to their oxygenation status and subsequent clinical events.Patient groups: Following informed consent blood samples were obtained from 20 children with SCD who were enrolled in an investigation of sleep hypoxia. After an awake blood oxgygen saturation by pulse oximetry (SaO2), an overnight recording was performed and a histogram generated showing proportions of time spent at various SaO2s. On the basis of mean sleeping SaO2s, the study population was divided into Group I (Normoxia, sleeping SaO2>94%, n=10) and Group II (Hypoxia, sleeping SaO2 <94%, n=10).Methods: VWF antigen level (vWF antigen) was determined by a latex enhanced immunoassay and Ristocetin Cofactor activity (RCof) was evaluated by platelet aggregometry. VWF multimer analysis was performed on SDS- 1.6% agarose gels and visualized using a polyclonal rabbit anti-human VWF antibody (Dako) followed by chemiluminescent detection using horse radish peroxidase-conjugated anti-rabbit IgG (Amersham). The presence of high molecular weight VWF multimers had to fulfill the following three criteria: (a) vWF multimers larger than the largest vWF multimers present in pooled normal plasma;(b)vWF multimers similar to the largest multimers present in a post-DDAVP infusion plasma sample analyzed on the same gel;(c) assessment by three independent investigators. Student's t-test was performed to compare the mean values of vWFAg, vWF:RCof, and vWF RCof:Ag ratio of Gps I and II.Results:We observed significant differences between normoxic vs. hypoxic children with SCD as depicted in the table below where results are shown as Mean values(1SD).vWF antigen levels correlated inversely (r2=0.28) and vWF RCof:Ag ratios correlated positively (r2=0.2) with sleeping SaO2s.vWF:Ag*vWF RCofvWF RCof:Ag Ratios**HMW-vWFGroup I >/94%120.8(30.9)116.7 (36.8)0.96(0.1)0/7 (performed to date)Group II <94%192.2 (69.4)147.3 (71.3)0.77(0.14)4/5 (performed to date)*p <0.003; **p <0.006Conclusions: Children with SCD and sleep hypoxia showed abnormalities in their vWF profiles when compared to normoxic children with SCD including elevated vWF antigen and qualitatively increased HMW multimers suggestive of hypoxia-mediated endothelial response. In addition, there is an unexpected discordance between these findings and decreased vWF RCof:Ag ratios. Since children with SCD and nocturnal hypoxemia have an increased rate of pain-associated vasoocclusion and CNS complications, our findings of vWF-related perturbations could provide an explanation for hypoxia-induced modulation of the proadhesive and prothrombotic state in these children.

Post‐appendectomy thrombotic thrombocytopenic purpura: A case report and review of the literature

Thrombotic thrombocytopenic purpura (TTP) is a syndrome characterized by microangiopathic hemolytic anemia and thrombocytopenia with varying degrees of renal dysfunction, neurologic signs and symptoms, and fever. Evidence has supported that a large proportion of cases of acquired TTP are due to the accumulation of ultralarge von Willebrand factor (vWF) multimers due to an acquired deficiency in the vWF cleaving protease, ADAMTS-13. TTP is rare in the post-surgical setting but is best described after cardiothoracic and vascular surgeries. We present a case of postoperative TTP first presenting with microangiopathic hemolytic anemia and thrombocytopenia 9 days after emergent appendectomy for a ruptured appendix. ADAMTS-13 and factor H levels returned normal and an ADAMTS-13 inhibitor was not identified. To our knowledge, this is the first case report of postoperative TTP after an appendectomy and the first report with correlative ADAMTS-13 data. Plasma exchange with fresh frozen plasma followed by cryopoor plasma, along with steroids resulted in eventual remission of TTP in our patient. Early postoperative diagnosis and aggressive management with consideration of initiation of plasma exchange is imperative to decrease the morbidity and morality associated with TTP.

Haemolytic uraemic syndrome and thrombotic thrombocytopenic purpura in southern Iran

In the first 2006 issue, Karimi et al.1 presented the results of a retrospective analysis conducted in 136 patients from southern Iran affected by thrombotic thrombocytopenic purpura (TTP) or haemolytic uraemic syndrome (HUS). Patients discharged during a 13-year period (April 1991March 2004) from the three main referral hospitals of the area were identified by review of medical records. TTP and HUS are two related disorders recognized more than 50 years ago. The hallmark of these disorders is represented by the concomitant occurrence of microangiopathic haemolytic anaemia, manifested by red-cell fragmentation, and thrombocytopenia. In addition, a wide spectrum of organ involvement with functional failure due to systemic microvascular arterial thrombosis is present in both syndromes, with the central nervous system more frequently and severely affected in TTP and the kidney in HUS. Recently, major advances have been made in the understanding of the pathophysiology of these disorders and in clarifying the basis of their clinical distinctive features2. TTP, once an ominous disease in more than 80-90% of cases, can now be effectively managed with intensive plasma exchange with a cure rate of 90% or more3. This is a heterogeneous disorder including possibly more than a single entity. The prototypical vascular lesions of TTP are characterised by platelet-rich thrombi within or beneath a damaged or swollen endothelium. Microvascular arterial thrombosis of TTP, leading to clamping and consumption of platelets, is triggered by ultra large von Willebrand factor (VWF) multimers released by insulted endothelial cells. Mechanical destruction of the red blood cells occurs when these cells are forced to circulate in small vessels obstructed by thrombi made of platelet, VWF and fibrin in high shear stress strictures. HUS occurs mostly in children, and it is characterised by variably severe renal disease that can lead to end-stage renal failure. In most cases, HUS is triggered by an infection caused by Shiga toxin producing Escherichia coli (O157:H7) usually manifested by watery or bloody diarrhoea. The disorder presents often with an acute onset. The clinical course may be self-limited despite no treatment and efficacy of plasma exchange is not proven. In both diseases an initiating condition is supposed to trigger the illness only in predisposed individuals. Clear examples of the importance of the individual genetic background in TTP are represented by the rare cases of homozygous deficiency of ADAMTS13, a recently discovered matalloprotease that is required to process the ultra large VWF multimers into smaller, less active species. In these patients TTP presents mainly as a familial or chronic relapsing disease. Severe reduction of ADAMTS13 is indeed also observed in a significant proportion (50 to 100%) of acquired idiopathic cases, indicating the critical role of ultra large VWF multimers in the pathogenesis of the disorder. In most, but not all cases with reduced ADAMTS13 activity, neutralizing autoantibodies have been demonstrated. Anyway, the diagnosis remains based on clinical features, and the clinical usefulness of dosing ADAMTS13 activity remains uncertain. Genetic predisposition to HUS is exemplified by the demonstration that an increasing number of non-Shiga toxin-associated cases occurs in patients with mutations in genes encoding complement regulatory proteins. More than 50 different mutations with variable penetrance have been described so far. Recently, among 156 Caucasian cases of familial or sporadic, non-Shiga-associated HUS, mutations have been found in 30 of them, including children and adults4. Triggering conditions were not always identifiable, although infection, pregnancy or drugs (cyclosporine, ticlopidine, clopidogrel, quinine, etc.) could be implicated in a significant proportion. Similar triggering factors have been implicated also in cases of typical TTP. VWF has not a critical role in the pathogenesis of microthrombi found in HUS, which are more rich in fibrin than in platelets and no ultra large VWF multimers are observed. This is in keeping with no or less severe deficiency of ADAMTS13 in HUS. Karimi et al.1 could not investigate the genetic individual predisposing factors in their patients, but they were able to extrapolate from medical records, distinctive cases of TTP or HUS, and to provide reliable relevant epidemiological data on the changing incidence rate of these disorders during the last 13 years. Although based on very simple criteria, the accuracy of differential diagnosis between 101 cases of HUS and the 35 cases of TTP finally identified seems confirmed by the clear-cut differences between the two Clinical letters

Unraveling the immunologic response in thrombotic thrombocytopenic purpura.

Unraveling the immunologic response in thrombotic thrombocytopenic purpura.

Von Willebrand factor, endothelial dysfunction, and cardiovascular disease.

von Willebrand factor (VWF), a glycoprotein involved in arterial thrombus formation, is released into the circulation by secretion from endothelial cells. Plasma VWF levels are determined by genetic factors including ABO blood groups and VWF mutations, and by non-genetic factors including aging, impaired nitric oxide production, inflammation, free radical production and diabetes. Plasma VWF levels have been proposed as a risk factor for cardiovascular events. Although they are only weakly associated with the risk of coronary heart disease (CHD) in the general population, they are a more promising CHD risk factor in high-risk populations with previous cardiovascular events, diabetes or old age. However, is it still unclear whether VWF levels directly determine the rate and severity of arterial thrombus formation or whether they merely reflect alteration in other endothelial functions. The future status of VWF levels as a cardiovascular risk factor depends on additional studies on the genetic determinants of both VWF levels and cardiovascular outcomes. Further studies on VWF levels as a predictor of the risk of stroke (rather than CHD) in elderly or other high-risk population are also promising. Such studies could lead to the clinical use of plasma VWF levels to refine the estimation of the cardiovascular risk and of the expected benefit of antithrombotic agents.

Thrombotic Thromboytopenic Purpura: Another Example of Immunomediated Thrombosis

Much progress has been made in recent years in understanding the mechanisms of TTP, through the demonstration of the role of ultra-large von Willebrand factor multimers and of ADAMTS13 deficiency in the pathogenesis of immune-mediated and genetic forms. There are, however, cases of TTP with normal or only slightly reduced levels of ADAMTS13, that may be due to the inhibition of the VWF cleaving activity ofADAMTS13 under the flow conditions of the microcirculation. The laboratory methods for assayingADAMTS13 are currently too complex to be used routinely on a large scale. In addition, they fail to reflect the conditions of blood flow that are necessary for the optimal interaction between VWF and its cleaving protease. The laboratory diagnosis of TTP is, therefore, still mainly clinical, being based on the presence of thrombocytopenia (due to increased intravascular consumption of blood platelets) and anemia (due to mechanical damage to red cells). There are still numerous questions to be answered. What are the pathogenic mechanisms of cases with normal or slightly reduced levels of ADAMTS13? What are the factors that periodically trigger the episodes of thrombocytopenia and anemia in the chronic recurrent forms, in the presence of constantly reduced levels of ADAMTS13? The behavior of anti-ADAMTS13autoantibodies following plasma exchange and therefore exposure to antigen (anamnestic response) is not well established. Likewise, the value of inducing immune tolerance by regular administrations of ADAMTS13, which will be possible when concentrates of the protease become available, has not been investigated.

The Role of ADAMT-13 in Platelet Adhesion in Flow: methods for Diagnosis of Thrombotic Thrombocytopenic Purpura

Von willebrand factor (VWF) is synthesized and released from endothelial cells and megakaryocytes. VWF multimers that are immobilized on the activated endothelial cells and unfolded by high shear stress, undergo proteolysis by specific VWF-cleaving protease, ADAMTS-13. Severe deficiency of ADAMTS-13 is followed by accumulation of ultralarge VWF multimers and development of thrombotic thrombocytopenic purpura (TTP). Several methods for detection of ADAMTS-13 activity are proposed. However, all these assays are lacking the condition of shear stress, in which rapid cleavage of VWF multimeric strings by the ADAMTS-13 occurs. Impact- R method testing the effect of plasma on normal platelet adhesion under shear stress allows rapid screening of acute TTP and discrimination from other thrombotic microangiopathies.

Von Willebrand Factor-Cleaving Protease (ADAMTS13) in the Course of Stem Cell Transplantation

Transplantation-associated microangiopathy (TAM) is a severe complication of stem cell transplantation. Although TAM shares many features with idiopathic thrombotic thrombocytopenic purpura or hemolytic uremic syndrome, the prognosis of TAM is worse. Clinical similarities and the observation that uncleaved ultralarge von Willebrand factor (ULVWF) multimers are found in the circulation of patients suffering from TAM suggest a defect in VWF proteolysis that may be due to a deficiency in ADAMTS13 activity. In this study the course of 28 consecutive patients, who received an allogeneic stem cell transplant was correlated to ADAMTS13 activity. Before stem cell transplantation, mean ADAMTS13 activity was within normal range. Within the first 8 weeks, mean activity declined to less than half the baseline activity. Furthermore, most of the patients showed normalization of ADAMTS13 activity. Low levels of ADAMTS13 activity were not correlated with clinical signs of thrombotic microangiopathy. However, two patients with clinical TAM had the lowest activity of all patients when suffering a severe bout of microangiopathy. Plasma exchange was not able to normalize ADAMTS13 deficiency in these patients, suggesting inactivation or consumption of ADAMTS13 activity in TAM.

Platelet Activation in Hemolytic Uremic Syndrome

Platelet consumption in platelet-fibrin aggregates leading to thrombocytopenia and small vessel obstruction are major features of the hemolytic uremic syndrome (HUS). Although thrombocytopenia has been correlated to poor prognosis, the mechanisms by which thrombocytopenia develops in HUS have not been completely elucidated. However, plausible explanations have been platelet contact with thrombogenic surfaces and/or direct contact with an aggregating agent. This article summarizes several mechanisms of platelet activation, interactions with leukocytes, chemokine release, complement activation, and antimicrobial defense. Specific mechanisms are outlined by which platelets may be activated, leading to thrombocytopenia during HUS. In diarrhea-associated HUS Shiga toxin has been shown to injure the endothelium, thus exposing the subendothelium, releasing tissue factor, and rendering the vessel wall prothrombotic. Shiga toxin also binds to and activates platelets. The toxin may activate endothelial cells and platelets simultaneously. In atypical HUS the alternative complement pathway is activated because of mutations in complement regulatory proteins. Mutated factor H does not bind to endothelium and platelets efficiently, enabling complement activation on these cells. In thrombotic thrombocytopenic purpura, intravascular platelet clotting occurs due to dysfunction of the von Willebrand factor (VWF) -cleaving protease ADAMTS13. Thrombi are formed by binding of platelets to ultralarge VWF multimers.

Identifying type Vicenza von Willebrand disease

Increased clearance of von Willebrand factor (VWF) is one of the main features of type Vicenza von Willebrand disease (VWD), a variant with plasma and platelet VWF level discrepancies and unusually large VWF multimers. Diagnosing type Vicenza VWD may not be easy, due to its heterogeneous phenotype. Here we describe the criteria we adopted to identify type Vicenza in a large group of VWD patients. Emphasizing the contribution of platelet VWF by comparison with plasma values, a first step involved selecting the candidate Vicenza patients on the basis of low or very low plasma VWF and a normal platelet VWF content. After excluding type 2A and 2B VWD patients, who may have normal platelet VWF, 18 candidates were found to meet our selection criteria. Genetic analysis revealed that 15 patients (from 5 unrelated families) were type Vicenza VWD and that all carried both G2220A and G3614A type Vicenza mutations barring one, who only had the G3614A mutation. All patients had a reduced VWF survival, and all but the patient with the G3614A mutation alone had ultralarge VWF multimers. Thus, low-plasma VWF associated with a normal platelet VWF content may be a first useful indicator for identifying type Vicenza VWD patients.

Thrombospondin-1 controls vascular platelet recruitment and thrombus adherence in mice by protecting (sub)endothelial VWF from cleavage by ADAMTS13

AbstractThe function of thrombospondin-1 (TSP-1) in hemostasis was investigated in wild-type (WT) and Tsp1-/- mice, via dynamic platelet interaction studies with A23187-stimulated mesenteric endothelium and with photochemically injured cecum subendothelium. Injected calcein-labeled WT platelets tethered or firmly adhered to almost all A23187-stimulated blood vessels of WT mice, but Tsp1-/- platelets tethered to 45% and adhered to 25.8% of stimulated Tsp1-/- vessels only. Stimulation generated temporary endothelium-associated ultralarge von Willebrand factor (VWF) multimers, triggering platelet string formation in 48% of WT versus 20% of Tsp1-/- vessels. Injection of human TSP-1 or thrombotic thrombocytopenic purpura (TTP) patient-derived neutralizing anti-ADAMTS13 antibodies corrected the defective platelet recruitment in Tsp1-/- mice, while having a moderate effect in WT mice. Photochemical injury of intestinal blood vessels induced thrombotic occlusions with longer occlusion times in Tsp1-/- venules (1027 ± 377 seconds) and arterioles (858 ± 289 seconds) than in WT vessels (559 ± 241 seconds, P < .001; 443 ± 413 seconds, P < .003) due to defective thrombus adherence, resulting in embolization of complete thrombi, a defect restored by both human TSP-1 and anti-ADAMTS13 antibodies. We conclude that in a shear field, soluble or local platelet-released TSP-1 can protect unfolded endothelium-bound and subendothelial VWF from degradation by plasma ADAMTS13, thus securing platelet tethering and thrombus adherence to inflamed and injured endothelium, respectively.

ADAMTS-13 regulates platelet adhesion under flow

ADAMTS-13 cleaves large and ultra-large von Willebrand factor multimers normally secreted by endothelial cells. Severe deficiency of this enzyme leads to thrombotic thrombocytopenic purpura (TTP). We applied the Impact-R system [Cone and plate(let) Analyzer, CPA] to determine optimal conditions for ADAMTS-13 function, to assess it's activity in TTP patients and to distinguish inherited TTP (inTTP) from acquired TTP (acTTP). The role of ADAMTS-13 in platelet adhesion under different conditions was investigated applying recombinant forms of VWF and ADAMTS-13. rVWF was first treated by rADAMTS-13 either in solution or when immobilized on the surface of the well, under static or flow conditions, in the presence or absence of BaCl2. The resulting cleaved VWF fragments were then immobilized and served to assess type 3 von Willebrand disease whole blood platelet adhesion under flow. Maximal effect of the rADAMTS-13 (decrease of platelet adhesion in the absence compared to the presence of BaCl2), was observed when the rVWF was pre-immobilized and the cleavage step took place under flow (85%). Mixing plasma ofTTP patients with normal blood (1:3) yielded a 1.6- to 2-fold increase of platelet adhesion under flow compared to mixing normal plasma with normal blood, at shear rate range of 1,800 - 2,500 s(-1) . Maximal increase of platelet adhesion was observed under 2,050 s(-1) . Under these conditions, the extent of adhesion was similarly higher in patients with inTTP and acTTP versus control [surface coverage (SC) 14.5 +/- 2.8% and 14.6 +/- 2.5% vs.7.4 +/- 1.7%, respectively]. ADAMTS-13 activity measured by collagen-binding test was similarly low (4.2 +/- 3.8% and 3.5 +/- 2.4% vs. 72.2 +/- 8.0%, respectively). An inverse correlation between SC and ADAMTS-13 activity was observed in a patient with inTTP assayed eight times during plasma infusion treatment. Addition of BaCl2 to the mixture of TTP plasma and normal whole blood yielded a decrease in platelet adhesion in inTTP (by 51%) but not in acTTP. The lack of reduction of platelet adhesion in acTTP could presumably be due to the presence of ADAMTS-13 inhibitor in these patients. These results suggest that VWF immobilization and high shear flow yielded optimal conditions for ADAMTS-13 activity, and that introduction of BaCl2 in the Impact-R (CPA) test may be useful for differentiation between inherited and acquired TTP.

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.

Cleavage of ultralarge multimers of von Willebrand factor by C-terminal–truncated mutants of ADAMTS-13 under flow

A disintegrin-like and metalloprotease with thrombospondin type 1-motif 13 (ADAMTS-13) cleaves the A2 domain of von Willebrand factor (VWF), converting the ultralarge (UL) and hyperactive VWF multimers freshly released from endothelial cells to smaller and less active forms found in plasma. Recombinant ADAMTS-13 lacking the C-terminal region is active under static conditions, but its functions under flow conditions have not been determined. Here, we show that VWF-cleaving activity measured under flow was preserved in an ADAMTS-13 mutant lacking the second to eighth thrombospondin-1 motifs and the complement components C1r/C1s, Uegf sea urchin fibropellins, and bone morphogenic protein 1 (CUB) domains, but was severely deficient in a mutant that was further truncated to remove the spacer domain. We also show that the mutant lacking the TSP-1 and CUB domains was hyperactive under flow, suggesting that the C-terminal region may negatively regulate ADAMTS-13 activity. The wild type and the mutant without the spacer were more active in the presence of plasma, raising the possibility of ADAMTS-13 cofactors in plasma.

Shear stress and the role of high molecular weight von Willebrand factor multimers in thrombus formation

High molecular weight von Willebrand factor (VWF) multimers have an important role in the formation of platelet thrombi under conditions of high shear stress resulting from rapid blood flow. Laboratory studies conducted using an endothelial cell system have shown that ultra-large VWF multimers (ULVWF) attach to the surface of histamine-stimulated endothelial cells and form large string-like structures to which platelets adhere. Platelet attachment is mediated through the interaction of the glycoprotein (Gp) Ibalpha subunit of the platelet GpIb-IX-V complex with the A1 domain of VWF. These platelet-ULVWF strings are rapidly cleaved by the addition of normal human plasma or the purified plasma metalloprotease ADAMTS-13 (a disintegrin and metalloprotease with thrombospondin type I motifs domains). Cleaving activity is absent from the plasma of patients with the congenital or acquired forms of thrombotic thrombocytopenic purpura (TTP) and from mixtures of normal plasma with plasma from patients with acquired TTP. The interaction of VWF with P-selectin is at least partially responsible for the attachment of the VWF strings to the endothelium. Increased tensile stress on ULVWF multimers resulting from the tethering of ULVWF strings to the endothelial surface by P-selectin in flowing blood may expose the cleavage site on the A2 domain of VWF, facilitating multimer degradation by ADAMTS-13. These studies have important implications in bleeding and thrombotic disorders, including von Willebrand disease and TTP.

Transcriptional regulation of ADAMTS13

The metalloproteinase ADAMTS13 cleaves VWF multimers instantaneously when they are released from endothelial cells. Absent or manifestly diminished proteolytic activity of ADAMTS13 results in the appearance and accumulation of ultralarge VWF multimers (ULVWFM) in plasma, characterised by the manifestation of Thrombotic Thrombocytopenic Purpura (TTP). Despite congenital defects, infections and the actions of drugs such as cyclosporine A, doxycycline and corticosteroids apparently are involved in its development. To examine the possibility of transcriptional regulation of ADAMTS13 activity, we analyzed RNA levels in various cell culture systems and the response to known and assumed modulators of gene expression. We demonstrate the expression of ADAMTS13 in liver homogenates and a parenchyma liver cell culture system Hep3B, supporting the hypothesis that liver is an important source of plasma ADAMTS13, whereas there was no alteration in gene expression after stimulation of liver cells with proinflammatory stimuli such as endotoxin, TNF-alpha, IL-6, IL-1beta as well as immuno-suppressive agents, such as cyclosporine A, a variety of steroids as well as doxycycline. Therefore, we analysed the ADAMTS13 gene for binding sites of transcription factors in silico and compared the data with those found in two sets of 24 genes considered either as differentially regulated by prototypic inflammatory regulation or as unvaried under various conditions. On the basis of these data, the promotor of ADAMTS13 features the characteristics of a gene, which remains unvaried under a variety of conditions. To our knowledge, the current data demonstrate for the first time, that an alteration in transcriptional activity is negligible in accounting for diminished proteolytic activity as observed under various experimental and, in particular, clinical conditions.

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.

Evaluation of ADAMTS-13 Activity and VWF:Ag Levels in Patients with Ischemic Stroke.

Background: The role of von Willebrand factor cleaving protease (ADAMTS-13) in the pathogenesis of ischemic stroke is still undefined. ADAMTS-13 cleaves ultra-large multimers of von Willebrand factor (ULVWF), and is believed to regulate the size thus the activity of VWF, especially on sites of high shear stress. By assuming the hypothesis, that a deficiency of ADAMTS-13 influences the thrombotic tendency such as intravascular aggregation and platelet thrombus formation, we analysed adult patients with onset of cerebral ischemia.Methods and Patients: 158 unrelated patients (female:89 /male:69) with an objectively confirmed ischemic stroke (n=126) or TIA (n=32), median age at first onset:42 years (range:17–71 years), and 82 healthy subjects, median age:28 years (range:16–52 years), were studied. In our study group we analysed on the one hand 95 patients (pts) with early onset of stroke/TIA <45 years (median age:32 years), and on the other hand 63 elderly pts >45 years (median age:54 years). None of the pts enrolled had overt evidence of autoimmune or malignancy disease. ADAMTS13 was measured with an assay based on the positive correlation between multimeric size and Ristocetin Cofactor activity of the VWF.Results: In our population the ADAMTS-13 activity was in median 95% (range= 40–185%) significantly higher than 91% (range=52–150%) among controls (p=0.002; OR 0.54). In the subgroup analysis we found no statistical differences in the mean ADAMTS-13 activities among pts with early onser (99% vs 91%; p=0.76, OR 0.93), and among pts with onset at age 45 years as compared to healthy controls (88% vs 91%; p=0.30, OR 1.25). Furthermore, we found statistical differences in the mean VWF:Ag levels among all pts as compared to healthy controls (135% vs 110%; p=0.01, OR 0.63), and in the elderly pts (140% vs 110%; p=0.01, OR 1.63). The young pts showed no statistical differences in the mean VWF:Ag levels compared with the control group (127% vs 110%; p=0.98, OR 0.99).Conclusion: The results from this study show that higher levels of ADAMTS-13 activity and VWF:Ag as compared to healthy controls are associated with ischemic stroke/TIA. Only in the elderly pts we observed a tendency to a negative association between ADAMTS-13 and VWF (lower ADAMTS-13 levels and higher levels of VWF:Ag). It may be, that higher ADAMTS-13 levels have a relevance to prevent thrombotic events.Further studies are needed to confirm whether the relationship between VWF and ADAMTS-13 plays an additional role in the pathogenesis on the onset of ischemic stroke.