Nele Vandeputte

Shielding of the A1 domain by the D'D3 domains of von Willebrand factor modulates its interaction with platelet glycoprotein Ib-IX-V

Soluble von Willebrand factor (VWF) has a low affinity for platelet glycoprotein (GP) Ibα and needs immobilization and/or high shear stress to enable binding of its A1 domain to the receptor. The previously described anti-VWF monoclonal antibody 1C1E7 enhances VWF/GPIbα binding and recognizes an epitope in the amino acids 764–1035 region in the N-terminal D′D3 domains. In this study we demonstrated that the D′D3 region negatively modulates the VWF/GPIb-IX-V interaction; (i) deletion of the D′D3 region in VWF augmented binding to GPIbα, suggesting an inhibitory role for this region, (ii) the isolated D′D3 region inhibited the GPIbα interaction of a VWF deletion mutant lacking this region, indicating that intramolecular interactions limit the accessibility of the A1 domain, (iii) using a panel of anti-VWF monoclonal antibodies, we next showed that the D′D3 region is in close proximity with the A1 domain in soluble VWF but not when VWF was immobilized; (iv) destroying the epitope of 1C1E7 resulted in a mutant VWF with an increased affinity for GPIbα. Our results support a model of domain translocation in VWF that allows interaction with GPIbα. The suggested shielding interaction of the A1 domain by the D′D3 region then becomes disrupted by VWF immobilization.

ADAMTS-13 plasma level determination uncovers antigen absence in acquired thrombotic thrombocytopenic purpura and ethnic differences.

Summary.  Background: The recently discovered plasma enzyme ADAMTS‐13 cleaves the A2‐domain of von Willebrand factor (VWF). A defective cleaving protease results in unusually large VWF multimers, which cause thrombotic thrombocytopenic purpura (TTP). Aim: Analysis of the ADAMTS‐13 antigen levels in TTP patients compared with normal donors. Methods: An antigen ELISA test was built, based on high affinity anti‐ADAMTS‐13 monoclonal antibodies, which were generated using genetic immunization. Results: Specificity of the ADAMTS‐13 antigen test was confirmed, as (i) plasma from a patient with acquired TTP but presenting without inhibitor did not contain antigen and (ii) the binding of recombinant ADAMTS‐13 was inhibited by increasing amounts of normal plasma. The assay is sensitive as it can detect antigen levels as low as 1.6% of normal. The concentration in normal pooled human plasma was determined (1.03 ± 0.15 μg mL−1) and arbitrarily set to 1 U mL−1. The antigen levels in congenital TTP samples (34 ± 21 mU mL−1, n = 2), as well as in samples from patients with acquired TTP (231 ± 287 mU mL−1, n = 11), were clearly reduced when compared with normal Caucasian donors (951 ± 206 mU mL−1, n = 16). Remarkably, normal Chinese donors have a significantly lower antigen titer (601 ± 129 mU mL−1, n = 15), when compared with normal Caucasians. Conclusions: Our results show that acquired TTP patients suffer mainly from ADAMTS‐13 antigen depletion, thereby indicating the importance of ADAMTS‐13 antigen determination in diagnosis and patient follow‐up.

Thrombotic thrombocytopenic purpura directly linked with ADAMTS13 inhibition in the baboon ( Papio ursinus )

Thrombotic thrombocytopenic purpura (TTP) is the prototypical microangiopathy characterized by disseminated microthromboses, hemolytic anemia, and ultimately organ dysfunction. A link with deficiency of the von Willebrand factor-cleaving protease (ADAMTS13) has been demonstrated, but additional genetic and/or environmental triggers are thought to be required to incite acute illness. Here we report that 4 days of ADAMTS13 functional inhibition is sufficient to induce TTP in the baboon (Papio ursinus), in the absence of inciting triggers because injections with an inhibitory monoclonal antibody (mAb) consistently (n = 6) induced severe thrombocytopenia (< 12 × 10(9)/L), microangiopathic hemolytic anemia, and a rapid rise in serum lactate dehydrogenase. Immunohistochemical staining revealed the characteristic disseminated platelet- and von Willebrand factor-rich thrombi in kidney, heart, brain, and spleen but not lungs. Prolonged inhibition (14 days, n = 1) caused myocardial ischemic damage and asplenia but not death. Control animals (n = 5) receiving equal doses of a noninhibitory anti-ADAMTS13 mAb remained unaffected. Our results provide evidence for a direct link between TTP and ADAMTS13 inhibition and for a mild disease onset. Furthermore, we present a reliable animal model of this disease as an opportunity for the development and validation of novel treatment strategies.

Restoration of Plasma von Willebrand Factor Deficiency Is Sufficient to Correct Thrombus Formation After Gene Therapy for Severe von Willebrand Disease

Objective—Gene therapy for severe von Willebrand disease (vWD) seems an interesting treatment alternative with long-term therapeutic potential. We investigated the feasibility of targeting the liver for ectopic expression of physiologically active von Willebrand factor (vWF). Methods and Results—The capacity of transgene-encoded murine vWF to restore vWF function was studied in a mouse model of severe vWD after liver-specific gene transfer by hydrodynamic injection. By using a hepatocyte-specific &agr;1 antitrypsin promoter, a considerably higher and longer-lasting vWF expression was obtained when compared with a cytomegalovirus promoter, reaching maximum vWF plasma levels that are 10±1 times higher than the wild-type level. Liver-expressed vWF showed the full range of multimers, including the high molecular weight multimers, and restored factor VIII plasma levels, consistent with correction of the bleeding time 3 but not 7 days after gene transfer. Importantly, transgene encoded plasma vWF restored proper platelet adhesion and aggregation in a FeCl3 induced thrombosis model. Conclusions—High ectopic expression of transgene encoded plasma vWF can be obtained after gene transfer to the liver. Liver-expressed vWF was fully multimerized and able to restore proper platelet plug formation in severe vWD. The liver therefore seems an attractive target for gene therapy for severe vWD.

Inhibition of von Willebrand factor-platelet glycoprotein Ib interaction prevents and reverses symptoms of acute acquired thrombotic thrombocytopenic purpura in baboons

The pathophysiology of thrombotic thrombocytopenic purpura (TTP) can be explained by the absence of active ADAMTS13, leading to ultra-large von Willebrand factor (UL-VWF) multimers spontaneously interacting with platelets. Preventing the formation of UL-VWF-platelet aggregates therefore is an attractive new treatment strategy. Here, we demonstrate that simultaneous administration of the inhibitory anti-VWF monoclonal antibody GBR600 and the inhibitory anti-ADAMTS13 antibody 3H9 to baboons (prevention group) precluded TTP onset as severe thrombocytopenia and hemolytic anemia were absent in these animals. In addition, partial VWF inhibition was not enough to prevent thrombocytopenia, demonstrating the specificity of this therapeutic strategy. GBR600 treatment of baboons during acute TTP (treatment group) resulted in a rapid recovery of severe thrombocytopenia similar to the platelet count increases observed in TTP patients treated by plasma exchange. Baboons in the control group only injected with 3H9 developed early stages of TTP as previously described. Hence, inhibiting VWF-GPIb interactions is an effective way to prevent and treat the early symptoms of acquired TTP in baboons.

The distal carboxyterminal domains of murine ADAMTS13 influence proteolysis of platelet-decorated VWF strings in vivo

Summary.  Background: The multidomain metalloprotease ADAMTS13 regulates the size of von Willebrand factor (VWF) multimers upon their release from endothelial cells. How the different domains in ADAMTS13 control VWF proteolysis in vivo remains largely unidentified. Methods: Seven C‐terminally truncated murine ADAMTS13 (mADAMTS13) mutants were constructed and characterized in vitro. Their ability to cleave VWF strings in vivo was studied in the ADAMTS13−/− mouse. Results: Murine MDTCS (devoid of T2‐8 and CUB domains) retained full enzyme activity in vitro towards FRETS‐VWF73 and the C‐terminal T6‐8 (del(T6‐CUB)) and CUB domains (delCUB) are dispensable under these assay conditions. In addition, mADAMTS13 fragments without the spacer domain (MDT and M) had reduced catalytic efficiencies. Our results hence indicate that similar domains in murine and human ADAMTS13 are required for activity in vitro, supporting the use of mouse models to study ADAMTS13 function in vivo. Interestingly, using intravital microscopy we show that removal of the CUB domains abolishes proteolysis of platelet‐decorated VWF strings in vivo. In addition, whereas MDTCS is fully active in vivo, partial (del(T6‐CUB)) or complete (delCUB) addition of the T2‐8 domains gradually attenuates its activity. Conclusions: Our data demonstrate that the ADAMTS13 CUB and T2‐8 domains influence proteolysis of platelet‐decorated VWF strings in vivo.

Inactivation of ADAMTS13 by plasmin as a potential cause of thrombotic thrombocytopenic purpura

Summary.  Background: ADAMTS13 deficiency causes accumulation of unusually large von Willebrand factor molecules, which cross‐link platelets in the circulation or on the endothelial surface. This process of intravascular agglutination leads to the microangiopathy thrombotic thrombocytopenic purpura (TTP). Most TTP patients have acquired anti‐ADAMTS13 autoantibodies that inhibit enzyme function and/or clear it from the circulation. However, the reason for ADAMTS13 deficiency is not always easily identified in a subset of patients. Objectives: To determine the origin of ADAMTS13 deficiency in a case of acquired TTP. Methods: Western blotting of ADAMTS13 in plasmas from acute and remission phases was used. Results: The ADAMTS13 deficiency was not caused by mutations or (detectable) autoantibodies; however, an abnormal ADAMTS13 truncated fragment (100 kDa) was found in acute‐phase but not remission‐phase plasma. This fragment resulted from enzymatic proteolysis, as recombinant ADAMTS13 was also cleaved when in the presence of acute‐phase but not remission‐phase plasma. Inhibitor screening showed that ADAMTS13 was cleaved by a serine protease that could be dose‐dependently inhibited by addition of exogenous α2‐antiplasmin. Examination of the endogenous α2‐antiplasmin antigen and activity confirmed deficiency of α2‐antiplasmin function in acute‐phase but not remission‐phase plasma. To investigate the possibility of ADAMTS13 cleavage by plasmin in plasma, urokinase‐type plasminogen activator was added to an (unrelated) congenital α2‐antiplasmin‐deficient plasma sample to activate plasminogen. This experiment confirmed cleavage of endogenous ADAMTS13 similar to that observed in our TTP patient. Conclusion: We report the first acquired TTP patient with cleaved ADAMTS13 and show that plasmin is involved.

Linker regions and flexibility around the metalloprotease domain account for conformational activation of ADAMTS-13

Recently, conformational activation of ADAMTS‐13 was identified. This mechanism showed the evolution from a condensed conformation, in which the proximal MDTCS and distal T2‐CUB2 domains are in close contact with each other, to an activated, open structure due to binding with von Willebrand factor (VWF).

Inhibition of Thrombin-Activatable Fibrinolysis Inhibitor and Plasminogen Activator Inhibitor-1 Reduces Ischemic Brain Damage in Mice

Background and Purpose— Cerebral ischemia and reperfusion is associated with activation of the coagulation cascade and fibrin deposition in cerebral microvessels. Both thrombin-activatable fibrinolysis inhibitor (TAFI) and plasminogen activator inhibitor-1 (PAI-1) attenuate fibrinolysis and are therefore attractive targets for the treatment of ischemic stroke. Methods— TAFI and PAI-1 were inhibited by monoclonal antibodies in a mouse model of transient middle cerebral artery occlusion. Twenty-four hours after stroke, mice were neurologically scored, cerebral thrombotic burden was assessed, and brain infarct sizes were calculated. Results— Inhibition of TAFI or PAI-1 significantly decreased cerebral infarct sizes by 50% 24 hours after stroke. This reduction in cerebral damage was associated with a significant decrease in fibrin(ogen) deposition in the ischemic brain. Concurrently, functional recovery of the animals was improved. Interestingly, combined targeting of TAFI and PAI-1 using low, and by themselves inactive, doses of antibodies improved cerebral blood flow and reduced cerebral fibrin(ogen) deposition and infarct sizes by 50%. When dual treatment was delayed to 1 hour after the start of reperfusion, it still reduced brain injury; however, this was not statistically significant. Conclusions— Targeting of PAI-1 and TAFI is protective in an ischemic stroke model by attenuating fibrin(ogen) deposition, thereby improving reperfusion. Combined inhibition has a co-operative effect that could become useful in ischemic stroke therapy.

The novel ADAMTS13-p.D187H mutation impairs ADAMTS13 activity and secretion and contributes to thrombotic thrombocytopenic purpura in mice

Congenital thrombotic thrombocytopenic purpura (TTP) is characterized by mutations in the ADAMTS13 gene, which either impair protein secretion or influence ADAMTS13 (A Disintegrin‐like And Metalloprotease domain with ThromboSpondin type‐1 motif, member 13) activity. Phenotypic consequences of these mutations have not yet been evaluated in animal models for TTP.