Deletion of GARP on mouse regulatory T cells is not sufficient to inhibit the growth of transplanted tumors.

GARP is a transmembrane protein that presents latent TGF-ß1 on the surface of regulatory T cells (Tregs). Neutralizing anti-GARP monoclonal antibodies that prevent the release of active TGF-ß1, inhibit the immunosuppressive activity of human Tregs in vivo. In this study, we investigated the contribution of GARP on mouse Tregs to immunosuppression in experimental tumors. Unexpectedly, Foxp3 conditional garp knockout (KO) mice challenged orthotopically with GL261 tumor cells or subcutaneously with MC38 colon carcinoma cells did not show prolonged survival or delayed tumor growth. Also, the suppressive function of KO Tregs was similar to that of wild type Tregs in the T cell transfer model in allogeneic, immunodeficient mice. In conclusion, garp deletion in mouse Tregs is not sufficient to impair their immunosuppressive activity in vivo.

High and long-term von Willebrand factor expression after Sleeping Beauty transposon-mediated gene therapy in a mouse model of severe von Willebrand disease.

Essentials von Willebrand disease (VWD) is the most common inherited bleeding disorder. Gene therapy for VWD offers long-term therapy for VWD patients. Transposons efficiently integrate the large von Willebrand factor (VWF) cDNA in mice. Liver-directed transposons support sustained VWF expression with suboptimal multimerization. SUMMARY: Background Type 3 von Willebrand disease (VWD) is characterized by complete absence of von Willebrand factor (VWF). Current therapy is limited to treatment with exogenous VWF/FVIII products, which only provide a short-term solution. Gene therapy offers the potential for a long-term treatment for VWD. Objectives To develop an integrative Sleeping Beauty (SB) transposon-mediated VWF gene transfer approach in a preclinical mouse model of severe VWD. Methods We established a robust platform for sustained transgene murine VWF (mVWF) expression in the liver of Vwf-/- mice by combining a liver-specific promoter with a sandwich transposon design and the SB100X transposase via hydrodynamic gene delivery. Results The sandwich SB transposon was suitable to deliver the full-length mVWF cDNA (8.4 kb) and supported supra-physiological expression that remained stable for up to 1.5 years after gene transfer. The sandwich vector stayed episomal (~60 weeks) or integrated in the host genome, respectively, in the absence or presence of the transposase. Transgene integration was confirmed using carbon tetrachloride-induced liver regeneration. Analysis of integration sites by high-throughput analysis revealed random integration of the sandwich vector. Although the SB vector supported long-term expression of supra-physiological VWF levels, the bleeding phenotype was not corrected in all mice. Long-term expression of VWF by hepatocytes resulted in relatively reduced amounts of high-molecular-weight multimers, potentially limiting its hemostatic efficacy. Conclusions Although this integrative platform for VWF gene transfer is an important milestone of VWD gene therapy, cell type-specific targeting is yet to be achieved.

An open conformation of ADAMTS-13 is a hallmark of acute acquired thrombotic thrombocytopenic purpura.

Essentials Conformational changes in ADAMTS-13 are part of its mode-of-action. The murine anti-ADAMTS-13 antibody 1C4 discriminates between folded and open ADAMTS-13. ADAMTS-13 conformation is open in acute acquired thrombotic thrombocytopenic purpura (TTP). Our study forms an important basis to fully elucidate the pathophysiology of TTP. SUMMARY: Background Acquired thrombotic thrombocytopenic purpura (aTTP) is an autoimmune disorder characterized by absent ADAMTS-13 activity and the presence of anti-ADAMTS-13 autoantibodies. Recently, it was shown that ADAMTS-13 adopts a folded or an open conformation. Objectives As conformational changes in self-antigens play a role in the pathophysiology of different autoimmune diseases, we hypothesized that the conformation of ADAMTS-13 changes during acute aTTP. Methods Antibodies recognizing cryptic epitopes in the spacer domain were generated. Next, the conformation of ADAMTS-13 in 40 healthy donors (HDs), 99 aTTP patients (63 in the acute phase versus 36 in remission), 12 hemolytic-uremic syndrome (HUS) patients and 63 sepsis patients was determined with ELISA. Results The antibody 1C4 recognizes a cryptic epitope in ADAMTS-13. Therefore, we were able to discriminate between a folded and an open ADAMTS-13 conformation. We showed that ADAMTS-13 in HDs does not bind to 1C4, indicating that ADAMTS-13 circulates in a folded conformation. Similar results were obtained for HUS and sepsis patients. In contrast, ADAMTS-13 of acute aTTP patients bound to 1C4 in 92% of the cases, whereas, in most cases, this binding was abolished during remission, showing that the conformation of ADAMTS-13 is open during an acute aTTP episode. Conclusions Our study shows that, besides absent ADAMTS-13 activity and the presence of anti-ADAMTS-13 autoantibodies, an open ADAMTS-13 conformation is also a hallmark of acute aTTP. Demonstrating this altered ADAMTS-13 conformation in acute aTTP will help to further unravel the pathophysiology of aTTP and lead to improved therapy and diagnosis.

Amplified endogenous plasmin activity resolves acute thrombotic thrombocytopenic purpura in mice.

Essentials Plasmin is able to proteolyse von Willebrand factor. It was unclear if plasmin influences acute thrombotic thrombocytopenic purpura (TTP). Plasmin levels are increased during acute TTP though suppressed via plasmin(ogen) inhibitors. Allowing amplified endogenous plasmin activity in mice results in resolution of TTP signs. SUMMARY: Background Thrombotic thrombocytopenic purpura (TTP) is an acute life-threatening pathology, caused by occlusive von Willebrand factor (VWF)-rich microthrombi that accumulate in the absence of ADAMTS-13. We previously demonstrated that plasmin can cleave VWF and that plasmin is generated in patients during acute TTP. However, the exact role of plasmin in TTP remains unclear. Objectives Investigate if endogenous plasmin-mediated proteolysis of VWF can influence acute TTP episodes. Results In mice with an acquired ADAMTS-13 deficiency, plasmin is generated during TTP as reflected by increased plasmin-α2-antiplasmin (PAP)-complex levels. However, mice still developed TTP, suggesting that this increase is not sufficient to control the pathology. As mice with TTP also had increased plasminogen activator inhibitor 1 (PAI-1) levels, we investigated whether blocking the plasmin(ogen) inhibitors would result in the generation of sufficient plasmin to influence TTP outcome in mice. Interestingly, when amplified plasmin activity was allowed (α2-antiplasmin-/- mice with inhibited PAI-1) in mice with an acquired ADAMTS-13 deficiency, a resolution of TTP signs was observed as a result of an increased proteolysis of VWF. In line with this, in patients with acute TTP, increased PAP-complex and PAI-1 levels were also observed. However, neither PAP-complex levels nor PAI-1 levels were related to TTP signs and outcome. Conclusions In conclusion, endogenous plasmin levels are increased during acute TTP, although limited via suppression through α2-antiplasmin and PAI-1. Only when amplified plasmin activity is allowed, plasmin can function as a back-up for ADAMTS-13 in mice and resolve TTP signs as a result of an increased proteolysis of VWF.

Platelet sequestration and activation during GalTKO.hCD46 pig lung perfusion by human blood is primarily mediated by GPIb, GPIIb/IIIa, and von Willebrand Factor.

BACKGROUND: Here, we ask whether platelet GPIb and GPIIb/IIIa receptors modulate platelet sequestration and activation during GalTKO.hCD46 pig lung xenograft perfusion. METHODS: GalTKO.hCD46 transgenic pig lungs were perfused with heparinized fresh human blood. Results from perfusions in which αGPIb Fab (6B4, 10 mg/l blood, n = 6), αGPIIb/IIIa Fab (ReoPro, 3.5 mg/l blood, n = 6), or both drugs (n = 4) were administered to the perfusate were compared to two additional groups in which the donor pig received 1-desamino-8-d-arginine vasopressin (DDAVP), 3 µg/kg (to pre-deplete von Willebrand Factor (pVWF), the main GPIb ligand), with or without αGPIb (n = 6 each). RESULTS: Platelet sequestration was significantly delayed in αGPIb, αGPIb+DDAVP, and αGPIb+αGPIIb/IIIa groups. Median lung "survival" was significantly longer (>240 vs. 162 min reference, p = 0.016), and platelet activation (as CD62P and ßTG) were significantly inhibited, when pigs were pre-treated with DDAVP, with or without αGPIb Fab treatment. Pulmonary vascular resistance rise was not significantly attenuated in any group, and was associated with residual thromboxane and histamine elaboration. CONCLUSIONS: The GPIb-VWF and GPIIb/IIIa axes play important roles in platelet sequestration and coagulation cascade activation during GalTKO.hCD46 lung xenograft injury. GPIb blockade significantly reduces platelet activation and delays platelet sequestration in this xenolung rejection model, an effect amplified by adding αGPIIb/IIIa blockade or depletion of VWF from pig lung.

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

BACKGROUND: 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). OBJECTIVES: Identification of cryptic epitope/exosite exposure after conformational activation and of sites of flexibility in ADAMTS-13. METHODS: The activating effect of 25 anti-T2-CUB2 antibodies was studied in the FRETS-VWF73 and the vortex assay. Cryptic epitope/exosite exposure was determined with ELISA and VWF binding assay. The molecular basis for flexibility was hypothesized through rapid automatic detection and alignment of repeats (RADAR) analysis, tested with ELISA using deletion variants and visualized using electron microscopy. RESULTS: Eleven activating anti-ADAMTS-13 antibodies, directed against the T5-CUB2 domains, were identified in the FRETS-VWF73 assay. RADAR analysis identified three linker regions in the distal domains. Interestingly, identification of an antibody recognizing a cryptic epitope in the metalloprotease domain confirmed the contribution of these linker regions to conformational activation of the enzyme. The proof of flexibility around both the T2 and metalloprotease domains, as shown by by electron microscopy, further supported this contribution. In addition, cryptic epitope exposure was identified in the distal domains, because activating anti-T2-CUB2 antibodies increased the binding to folded VWF up to ~3-fold. CONCLUSION: Conformational activation of ADAMTS-13 leads to cryptic epitope/exosite exposure in both proximal and distal domains, subsequently inducing increased activity. Furthermore, three linker regions in the distal domains are responsible for flexibility and enable the interaction between the proximal and the T8-CUB2 domains.

Desmopressin treatment improves platelet function under flow in patients with postoperative bleeding.

BACKGROUND: Patients undergoing major cardiothoracic surgery are subjected to dilution, owing to massive fluid infusion and blood component transfusion. These patients may experience bleeding perioperatively, and are frequently treated with the endothelium-activating agent desmopressin. OBJECTIVES: To investigate the effect of desmopressin administration on von Willebrand factor (VWF)-dependent coagulant and platelet functions under flow conditions. PATIENTS/METHODS: Blood from 16 patients with postoperative bleeding was obtained before and after desmopressin treatment (0.3 µg kg(-1) body weight), and assessed for coagulant properties and platelet function. Furthermore, VWF antigen levels and multimer composition were determined in both samples. RESULTS: Desmopressin treatment did not change thrombin generation in plasma or whole blood thromboelasticity. Also coagulation factor levels (other than factor VIII) and coagulation times were unchanged, suggesting that desmopressin treatment did not have a major effect on the coagulant activity. On the other hand, desmopressin treatment raised the already high plasma levels of VWF from a median of 116 IU mL(-1) (interquartile range [IQR] 102-154 IU mL(-1) ) to a median of 160 IU mL(-1) (IQR 126-187 IU mL(-1) ) (P = 0.007), owing to accumulation of the high molecular weight VWF multimers. Furthermore, desmopressin treatment caused an increase in collagen-dependent thrombus formation and platelet phosphatidylserine exposure. Markers of thrombus formation correlated with the plasma levels of VWF. In vitro control experiments confirmed a major contribution of VWF to thrombus formation and procoagulant activity under conditions of blood dilution. CONCLUSIONS: Desmopressin treatment of patients with bleeding complications after cardiothoracic surgery induces the release of high molecular weight VWF multimers, which enhance platelet activation and thrombus formation under flow conditions.

Platelets from flowing blood attach to the inflammatory chemokine CXCL16 expressed in the endothelium of the human vessel wall.

Endothelial chemokine CXC motif ligand 16 (CXCL16) expression is associated with atherosclerosis, while platelets, particularly those attaching to atherosclerotic plaque, contribute to all stages of atherosclerotic disease. This investigation was designed to examine the role of CXCL16 in capturing platelets from flowing blood. CXCL16 was expressed in human atherosclerotic plaques, and lesion severity in human carotid endarterectomy specimens was positively correlated with CXCL16 levels. CXCL16 expression in plaques was co-localised with platelets deposited to the endothelium. Immobilised CXCL16 promoted CXCR6-dependent platelet adhesion to the human vessel wall, endothelial cells and von Willebrand factor during physiologic flow. At low shear, immobilised CXCL16 captured platelets from flowing blood. It also induced irreversible platelet aggregation and a rise in intra-platelet calcium levels. These results demonstrate that endothelial CXCL16's action on platelets is not only limited to platelet activation, but that immobilised CXCL16 also acts as a potent novel platelet adhesion ligand, inducing platelet adhesion to the human vessel wall.

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

BACKGROUND: 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. OBJECTIVES: To identify the in vitro effect of a novel ADAMTS13 mutation and to investigate whether this mutation induces TTP in vivo. METHODS: All 29 ADAMTS13 exons with exon-intron boundaries of a patient with pregnancy-onset TTP were sequenced. Wild-type and mutant ADAMTS13 proteins were both transiently and stably expressed in human embryonic kidney cells, and their activity was evaluated in vitro using fluorescence resonance energy transfer and flow assays. Molecular dynamics simulations were performed to study Ca(2+) stability. Adamts13(-/-) mice were hydrodynamically injected with wild-type and mutant expression plasmids and triggered with recombinant human von Willebrand factor. RESULTS: We identified a novel heterozygous c.559G>C mutation in exon 6 of the proposita's ADAMTS13 gene. This mutation resulted in a p.Asp187His substitution (p.D187H), which was located in the high affinity Ca(2+) -binding site in the metalloprotease domain of ADAMTS13. The homozygous p.D187H mutation down-regulated ADAMTS13 activity in vitro. Impaired proteolytic activity was linked to unstable Ca(2+) binding as visualized using a molecular dynamics simulation. In addition, the p.D187H mutation affects protein secretion in vitro. In Adamts13(-/-) mice, the homozygous p.D187H mutation reduced ADAMTS13 secretion and activity and contributed to TTP when these mice were triggered with recombinant human von Willebrand factor. CONCLUSIONS: Our data indicate that the p.D187H mutation impairs ADAMTS13 activity and secretion and is responsible for TTP onset in mice.

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

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.

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.

Identification of a VWF peptide antagonist that blocks platelet adhesion under high shear conditions by selectively inhibiting the VWF-collagen interaction.

BACKGROUND: Because the collagen-VWF-GPIb/IX/V axis plays an important role in thrombus formation, it represents a promising target for development of new antithrombotic agents. OBJECTIVES: We used phage display to identify potential small peptides that interfere with the VWF-collagen binding and might serve as lead products for the development of possible oral antithrombotic compounds. METHODS: A random linear heptamer peptide library was used to select VWF-binding peptides. RESULTS: We identified a phage clone, displaying the YDPWTPS sequence, further referred to as L7-phage, that bound to VWF in a specific and a dose-dependent manner. This L7-phage specifically inhibited the VWF-collagen interaction under both static and flow conditions. Epitope mapping using deletion mutants of VWF revealed that the L7-phage does not bind to the known collagen-binding A3 domain within VWF, but to the more carboxyterminal situated C domain. This inhibition was not due to steric hindrance of the A3 domain-collagen interaction by the L7-phage. Indeed, a tetrabranched multi-antigen peptide (MAP) presenting four copies of the peptide, but not the scrambled MAP, also inhibited VWF-collagen interaction under conditions of high shear stress at a concentration of 148 nmol L(-1). CONCLUSIONS: Based on these results, we conclude that we have identified the first peptide antagonist that binds to the VWF C domain and by this specifically inhibits the VWF binding to collagen, suppressing platelet adhesion and aggregation under high shear conditions. As a consequence, this peptide and its future derivates are potentially interesting antithrombotic agents.

Collagen-mimetic peptides mediate flow-dependent thrombus formation by high- or low-affinity binding of integrin alpha2beta1 and glycoprotein VI.

BACKGROUND: Collagen acts as a potent surface for platelet adhesion and thrombus formation under conditions of blood flow. Studies using collagen-derived triple-helical peptides have identified the GXX'GER motif as an adhesive ligand for platelet integrin alpha2beta1, and (GPO)(n) as a binding sequence for the signaling collagen receptor, glycoprotein VI (GPVI). OBJECTIVE: The potency was investigated of triple-helical peptides, consisting of GXX'GER sequences within (GPO)(n) or (GPP)(n) motifs, to support flow-dependent thrombus formation. RESULTS: At a high-shear rate, immobilized peptides containing both the high-affinity alpha2beta1-binding motif GFOGER and the (GPO)(n) motif supported platelet aggregation and procoagulant activity, even in the absence of von Willebrand factor (VWF). With peptides containing only one of these motifs, co-immobilized VWF was needed for thrombus formation. The (GPO)(n) but not the (GPP)(n) sequence induced GPVI-dependent platelet aggregation and procoagulant activity. Peptides with intermediate affinity (GLSGER, GMOGER) or low-affinity (GASGER, GAOGER) alpha2beta1-binding motifs formed procoagulant thrombi only if both (GPO)(n) and VWF were present. At a low-shear rate, immobilized peptides with high- or low-affinity alpha2beta1-binding motifs mediated formation of thrombi with procoagulant platelets only in combination with (GPO)(n). CONCLUSIONS: Triple-helical peptides with specific receptor-binding motifs mimic the properties of native collagen I in thrombus formation by binding to both platelet collagen receptors. At a high-shear rate, either GPIb or high-affinity (but not low-affinity) GXX'GER mediates GPVI-dependent formation of procoagulant thrombi. By extension, high-affinity binding for alpha2beta1 can control the overall platelet-adhesive activity of native collagens.

Multiple ways to switch platelet integrins on and off.

In the classical concept of platelet integrin activation, it is considered that unidirectional conformational changes of alpha(IIb)beta(3) and alpha(2)beta(1) regulate the adhesiveness of platelets for fibrin(ogen) and collagen, respectively. Here, we summarize recent evidence that these conformational changes: (i) can also occur in the reverse direction; and (ii) are not independent events. Platelet stimulation through the P2Y(12) receptors provokes only transient alpha(IIb)beta(3) activation via signaling routes involving phosphoinositide 3-kinases and Rap1b. Furthermore, alpha(IIb)beta(3) can be secondarily inactivated in platelets with prolonged high Ca(2+) rises, which expose phosphatidylserine and bind coagulation factors. Thus, platelet stimulation with strong agonists (collagen and thrombin) also results in transient integrin activation. Integrin alpha(2)beta(1) is found to be activated by a mechanism that is directly linked to alpha(IIb)beta(3) activation. Integrin alpha(2)beta(1) can adopt different activation states, depending on the trigger. Conclusively, reversibility and synchrony of platelet integrin activation are newly identified mechanisms to restrict thrombus growth and to allow optimal coagulation factor binding. Back-shifting of activated integrins towards their resting state may be a novel goal of antithrombotic medication.

Development of monoclonal antibodies that inhibit platelet adhesion or aggregation as potential anti-thrombotic drugs.

Cardiovascular disease is the major cause of mortality in Western countries. Platelets play a crucial role in the development of arterial thrombosis and other pathophysiologies leading to clinical ischemic events. In the damaged vessel wall, platelets adhere to the subendothelium through an interaction with von Willebrand factor (VWF), which forms a bridge between subendothelial collagen and the platelet receptor glycoprotein (GP) Ib/IX/V. This reversible adhesion allows platelets to roll over the damaged area, decreasing their velocity and resulting in strong platelet activation. This leads to the conformational activation of the platelet GPIIb/IIIa receptor, fibrinogen binding and finally to platelet aggregation. As each interaction (collagen-VWF, VWF-GPIb and GPIIb/IIIa-fibrinogen) plays an essential role in primary haemostasis, loss of either of these interactions results in a bleeding diathesis, implying that interfering with these interactions might result in an anti-thrombotic effect. Whereas GPIIb/IIIa antagonists indeed are effective anti-thrombotics, it has been suggested that drugs which block the initial steps of thrombus formation (collagen-VWF or VWF-GPIb interaction) might have advantages over the ones that merely inhibit platelet aggregation. In this review we will discuss and compare the development of monoclonal antibodies (moAbs) that inhibit platelet adhesion or platelet aggregation. The effect of the moAbs in in vitro experiments, in in vivo models and in clinical trials will be described. Benefits, limitations, current applications and the future perspectives in the development of antibodies for each target will be discussed.

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

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 microg 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.

Development of antibodies that interfere with the collagen-VWF-GPIb axis as new antithrombotics.

The epitope and the antithrombotic effect of 6B4, an antibody that inhibits GPIb, the receptor for von Willebrand Factor (VWF) on blood platelets, and of 82D6A3, an antibody against VWF that prevents the binding of VWF to collagen, were characterised. By using canine-human chimeras, alanine-scans, phage display, mutant analysis and modeling both the epitope of 6B4 in the N-terminal domain of GPIb, and of 82D6A3 in the VWF-A3 domain, could be mapped. As both epitopes furthermore are part of the ligand binding sites, this at once also explained the mechanism of the inhibition by the antibodies. Next both antibodies were tested in a thrombosis model in a stenosed artery in baboons, where they showed potent antithrombotic activities, without a noteworthy prolongation of the bleeding time. With this we thus could reveal two new strategies to prevent arterial thrombosis, which presumably may be safer than the currently available antiplatelet agents.

The von Willebrand factor self-association is modulated by a multiple domain interaction.

BACKGROUND: Platelet adhesion and aggregation at sites of vascular injury exposed to rapid blood flow require von Willebrand factor (VWF). VWF becomes immobilized by binding to subendothelial components or by a self-association at the interface of soluble and surface-bound VWF. OBJECTIVES: As this self-association has been demonstrated only under shear conditions, our first goal was to determine whether the same interaction could be observed under static conditions. Furthermore, we wanted to identify VWF domain(s) important for this self-association. RESULTS: Biotinylated VWF (b-VWF) interacted dose-dependently and specifically with immobilized VWF in an enzyme-linked immunosorbent assay (ELISA) assay, showing that shear is not necessary to induce the VWF self-association. Whereas anti-VWF monoclonal antibodies (mAbs) had no effect on the self-association, the proteolytic VWF-fragments SpII(1366-2050) and SpIII(1-1365) inhibited the b-VWF-VWF interaction by 70 and 80%, respectively. Moreover, a specific binding of b-VWF to immobilized Sp-fragments was demonstrated. Finally, both biotinylated SpII and SpIII were able to bind specifically to both immobilized SpII and SpIII. Similar results were observed under flow conditions, which confirmed the functional relevance of our ELISA system. CONCLUSION: We have developed an ELISA binding assay in which a specific VWF self-association under static conditions can be demonstrated. Our results suggest a multiple domain interaction between immobilized and soluble VWF.

The hemostatic system.

The hemostatic system comprises platelet aggregation, coagulation and fibrinolysis also termed primary, secondary and tertiary hemostasis. From the platelet transcriptome 6000 mRNA species and represent receptors, ion channels, signalling molecules, kinases, phosphatases, and structural, metabolic and regulatory proteins. This abundance of regulatory proteins points towards the importance of signal transduction in platelet function. First platelets adhere to collagen, this induces activation signals such as TXA(2) that induces further Ca(2+) increase. Consecutively, fibrinogen binds to the integrin alpha(IIb)beta(3) resulting in aggregation.This self-amplifying process is controlled by signals, from endothelial cells, to restrict the platelet plug to the site of vessel injury. Secondary hemostasis (coagulation) consists of an extrinsic and intrinsic pathway. Thrombin is generated via Factor Xa resulting from the extrinsic tenase reaction that is turned of by tissue factor pathway inhibitor. While thrombin generation is maintained via positive feedback mechanisms activating factors V, VIII and XI. Excess thrombin is inhibited by antithrombin or by autodownregulation via activation of protein C. Since minor injuries are common, platelets and plasma clotting factors constantly produce clots to stop bleeding. If clots remained after the tissue healing, the vascular bed would become obstructed with clots therefore this is regulated by fibrinolysis, tertiary hemostasis. Tissue-type plasminogen activator synthesised by the endothelium, converts plasminogen to plasmin, the clot lysis enzyme. Plasmin clears the blood vessels by degrading fibrin. Fibrinolysis is controlled by plasminogen activators inhibitor (PAI-1), alpha2-antiplasmin and alpha2-macroglobulin, and thrombin-activatable fibrinolysis inhibitor (TAFI).