ADAMTS13 Expressed In Platelets Offers Systemic Protection Against Arterial Thrombosis and Therapeutic Benefits In Murine Models Of Thrombotic Thrombocytopenic Purpura

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 446-446
Author(s):  
Brandy Pickens ◽  
Yingying Mao ◽  
X. Long Zheng
Keyword(s):  
Platelet Count ◽  
Transgenic Mice ◽  
Thrombotic Thrombocytopenic Purpura ◽  
Arterial Thrombosis ◽  
Oxidative Injury ◽  
Therapeutic Benefit ◽  
Full Length ◽  
Murine Models ◽  
Wild Type ◽  
Thrombocytopenic Purpura

ADAMTS13, a plasma metalloprotease, cleaves von Willebrand factor (VWF) and inhibits arterial thrombosis and inflammatory response. Deficiency of plasma ADAMTS13, due hereditary mutations in ADAMTS13 gene or autoantibodies against ADAMTS13 protease, results in a potentially fatal syndrome, thrombotic thrombocytopenic purpura (TTP). Plasma infusion or exchange is the only effective therapy available to date. To develop novel therapeutics against TTP, we tested a hypothesis that recombinant ADAMTS13, expressed specifically in platelets, may offer systemic protection against arterial thrombosis and therefore provide therapeutic benefit for TTP. To test this hypothesis, we first generated transgenic mice (JAX B6SJL) carrying a human full-length ADAMTS13 gene under a murine platelet glycoprotein 1b (CD41) promoter. The transgenic mice were then bred with Adamts13-/- (CAST/Ei) mice for >4 generations. By Western blotting and activity assay, we show that a full-length human ADAMTS13 protein (∼195 kDa) and its proteolytic activity toward a FRETS-hVWF73 peptide are detected in platelet lysate obtained from transgenic (rA13-PltTG) mice but not in Adamts13-/- mice or wild-type mice. Little to no ADAMTS13 activity was detected in plasma in transgenic mice, suggesting the expressed human ADAMTS13 is stored inside platelets. ADAMTS13 was releasable upon stimulation with thrombin (1 U/ml), collagen (10 µg/ml), and AYPGKF (0.5 mM). More significantly, rA13-PltTG mice had higher baseline platelet count than Adamts13-/- mice, but exhibited a dramatically reduced rate of thrombus formation in mesenteric arterioles after oxidative injury with 10% ferric chloride as compared with Adamts13-/-mice and wild-type mice. Finally, rA13-PltTG mice were protected from Shigatoxin-2 (Stx-2) or murine recombinant VWF (mVWF)-induced “TTP-like” syndrome, as demonstrated by fewer rA13-PltTG mice having thrombocytopenia (defined by a 40% drop in platelet count from the baseline after challenge with Stx-2 or mVWF), faster and more complete recovery of thrombocytopenia, and significantly higher survival rate than Adamts13-/- mice. In summary, we have generated transgenic mice expressing human ADAMTS13 in platelets. Platelet ADAMTS13 is releasable upon stimulation by agonists and biologically functional in proteolytic cleavage of VWF in vitro. The platelet-derived ADAMTS13 offers systemic protection against arterial thrombosis after oxidative injury and provide a therapeutic benefit to murine models of TTP, resulting from hereditary ADAMTS13 deficiency. We are now in the process testing the efficacy of this strategy as a novel therapeutic for acquired TTP with inhibitors. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Platelet-delivered ADAMTS13 inhibits arterial thrombosis and prevents thrombotic thrombocytopenic purpura in murine models

Blood ◽  
2015 ◽  
Vol 125 (21) ◽  
pp. 3326-3334 ◽  
Author(s):  
Brandy Pickens ◽  
Yingying Mao ◽  
Dengju Li ◽  
Don L. Siegel ◽  
Mortimer Poncz ◽  
...  
Keyword(s):  
Thrombotic Thrombocytopenic Purpura ◽  
Vascular Injury ◽  
Arterial Thrombosis ◽  
Murine Models ◽  
Thrombocytopenic Purpura ◽  
Key Points

Key Points Platelet-delivered ADAMTS13 inhibits arterial thrombosis after vascular injury. Platelet-delivered ADAMTS13 also prevents thrombotic thrombocytopenic purpura.

Pregnancy Does Not Affect The Thrombotic Thrombocytopenic Purpura (TTP) phenotype Of ADAMTS13-Deficient Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3558-3558
Author(s):  
Yael Hayon ◽  
David Varon ◽  
Yosef Kalish
Keyword(s):  
Platelet Count ◽  
Mouse Model ◽  
Thrombotic Thrombocytopenic Purpura ◽  
Peripheral Blood ◽  
Wild Type ◽  
Thrombocytopenic Purpura ◽  
Female Mice ◽  
Blood Smears ◽  
Peripheral Blood Smears ◽  
Deficient Mice

Abstract Thrombotic thrombocytopenic purpura (TTP) is a life threatening disease, characterized by sudden onset of the pentad of fever, hemolytic anemia, thrombocytopenia, renal dysfunction and neurological findings. Previous studies in ADAMTS13-deficient mice demonstrated a dependence on both genetic and environmental factors for the expression of the disease. Though homozygous Adamts13-/- mice on a C57BL6/J (B6) background are indistinguishable from wild-type controls, introduction of the CAST/EiJ genetic background results in a spontaneous TTP-like syndrome in a subset of mice. The aim of this study was to evaluate the effect of pregnancy on TTP expression in this mouse model of TTP. Adamts13 +/- mice were backcrossed for 20 generations to C57BL/6J to generate N20 B6 Adamts13+/- progeny. These mice were backcrossed for 2 generations into the CAST/EiJ mouse strain to generate N2F1 Adamts13-/- female mice that were tested and compared to N20F1 B6 Adamts13-/- female mice. Two groups of 20 Adamts13-/- female mice from each strain were tested. Mice from one group were mated with wild type males, and mice from the second group were housed separately from male mice. Pregnant Adamts13-/- mice were compared to control non-pregnant Adamts13-/- female mice. Mice were monitored for a full profile of blood parameters and peripheral blood smears weekly for 10 weeks. Data was collected for mortality, pregnancy outcome and litter size. Von Willebrand factor (VWF) levels were also measured. All Adamts13-/- mice in the B6 non-pregnant group appeared healthy and exhibited normal blood count parameters, without microangiopathic changes on peripheral blood smears. None of Adamts13-/- mice in the B6 pregnant group developed TTP. Mean platelet count in this group was 844,000±127,000 cells/µl compared to 666,000±56,000 cells/µl in the B6 non-pregnant group (p<0.01) In contrast to the B6 results, Adamts13-/- CAST/EiJ non-pregnant mice exhibited lower platelet counts 369,000±89,000 cells/µl with spontaneous TTP-like disease in 54% of mice, as expected from previous studies. Pregnancy did not worsen TTP expression in Adamts13-/- CAST/EiJ mice. Only 45% of mice in the CAST/EiJ pregnant group developed low platelet counts with a higher mean platelet count of 511,000±160,000 cells/µl, compared to non-pregnant group(p<0.01). The physiological rise in VWF levels of pregnancy had no effect on TTP susceptibility in this model. VWF antigen levels were 167%, 203%, 197% and 245% in B6 non-pregnant, B6 pregnant, CAST/EiJ non-pregnant and pregnant respectively, compared to wild-type B6 levels. Four mice died during follow-up, 2 in the B6 pregnant and 2 in the CAST/EiJ non-pregnant groups. In summary, TTP expression was unique to Adamts13-/- CAST/EiJ mice. Pregnancy and elevated VWF levels had no deleterious effect on TTP susceptibility in this mouse model. Disclosures: No relevant conflicts of interest to declare.

Molecular characterization of four ADAMTS13 mutations responsible for congenital thrombotic thrombocytopenic purpura (Upshaw-Schulman syndrome)

2007 ◽  
Vol 98 (09) ◽  
pp. 593-599 ◽  
Author(s):  
Antoine Hommais ◽  
Julie Rayes ◽  
Anne Houllier ◽  
Bernadette Obert ◽  
Paulette Legendre ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Thrombotic Thrombocytopenic Purpura ◽  
Proteolytic Activity ◽  
Specific Activity ◽  
Electrophoretic Analysis ◽  
Full Length ◽  
Wild Type ◽  
Thrombocytopenic Purpura ◽  
Congenital Thrombotic Thrombocytopenic Purpura

SummaryADAMTS13 mutations S203P, R268P, R507Q and A596V were previously identified in French patients with hereditary thrombotic thrombocytopenic purpura (TTP) (Upshaw-Schulman syndrome). Mutated recombinant (r) ADAMTS13 were transiently expressed in COS-7 cells and characterized in comparison with wild-type (WT) rADAMTS13.ADAMTS13 antigen was qualitatively and quantitatively estimated by electrophoretic analysis and ELISA. Enzymatic activity was qualitatively and quantitatively estimated using GST-VWF73,FRETS-VWF73 fragments and full-length rVWF-WT as substrates. The four mutants and rADAMTS13-WT were present within the cells. Secretion level of rADAMTS13-WT reached 1,200 ng/ml. The four mutations strongly altered the secretion and biological activity of rADAMTS13. The percentage secretion was 21, 38 and 17% for rADAMTS13-S203P, -R268P and -A596V compared with rADAMTS13- WT. rADAMTS13-R507Q concentration was under the detection limit of the assay. In the four cases, no enzymatic activity was detected. After concentration, we confirmed that mutations S203P and R268P totally abolished the proteolytic activity of ADAMTS13. Due to the very low protease concentration, activity of rADAMTS13-R507Q was below the threshold of the assays. rADAMTS13-A596V had no proteolytic activity towards the full-length rVWF-WT whereas it exhibited a decreased specific activity of about 30% of that of rADAMTS13- WT towards FRETS-VWF73 fragment. Binding study of mutated rADAMTS13-S203P, -R268P and -A596V showed that the three mutations strongly decreased the interaction of ADAMTS13 with VWF. In conclusion, the four mutations, which led to a secretion defect, a loss of enzymatic activity and a decreased binding to the substrate, are responsible for the hereditary TTP in patients.

Thrombotic Thrombocytopenic Purpura: Mechanism for Effectiveness of Plasmapheresis

1979 ◽  
Author(s):  
J. G. Kelton ◽  
P. B. Neame ◽  
I. Walker ◽  
A. G. Turpie ◽  
J. McBride ◽  
...  
Keyword(s):  
Platelet Count ◽  
Thrombotic Thrombocytopenic Purpura ◽  
The Other ◽  
Unknown Etiology ◽  
Normal Range ◽  
Antiplatelet Antibody ◽  
Thrombocytopenic Purpura ◽  
Serious Illness ◽  
Normal Platelet ◽  
Very High

Thrombotic thrombocytopenic purpura (TTP) is a rare but serious illness of unknown etiology. Treatment by plasmapheresis has been reported to be effective but the mechanism for benefit is unknown. We have investigated the effect of plasmapheresis in 2 patients with TTP by quantitating platelet associated IgG (PAIgG) levels prior to and following plasmapheresis. Both patients had very high levels of PAIgG at presentation (90 and A8 fg IgG/platelet respectively, normal 0-5). in both, the PAIgG levels progressively fell to within the normal range and the platelet count rose following plasmapheresis. One patient remained in remission with normal platelet counts and PAIgG levels. The other relapsed after plasmapheresis and the PAIgG level rose prior to the fall in platelet count. Plasmapheresis was repeated and resulted in normalization of both the platelet count and PAIgG level. It is suggested that plasmapheresis removes antiplatelet antibody or immune complexes which may be of etiological importance in this illness.

Thrombotic Thrombocytopenic Purpura: Mechanism for Effectiveness of Plasmapheresis

1979 ◽  
Author(s):  
J.G. Kelton ◽  
P.B. Neame ◽  
I. Walker ◽  
A.G. Turpie ◽  
J. McBride ◽  
...  
Keyword(s):  
Platelet Count ◽  
Thrombotic Thrombocytopenic Purpura ◽  
The Other ◽  
Unknown Etiology ◽  
Normal Range ◽  
Antiplatelet Antibody ◽  
Thrombocytopenic Purpura ◽  
Serious Illness ◽  
Normal Platelet ◽  
Very High

Thrombotic thrombocytopenic purpura (TTP) is a rare but serious illness of unknown etiology. Treatment by plasmapheresis has been reported to be effective but the mechanism for benefit is unknown. We have investigated the effect of plasmapheresis in 2 patients with TTP by guantitating platelet associated IgG (PAIgG) levels prior to and following plasmapheresis. Both patients had very high levels of PAIgG at presentation (90 and 48 fg IgG/platelet respectively, normal 0-5). In both, the PAIgG levels progressively fellto within the normal range and the platelet count rose following plasmapheresis. One patient remained in remission with normal platelet counts and PAIgG levels. The other relapsed after plasmapheresis and the PAIgG levelrose prior to the fall in platelet count. Plasmapheresis was repeated and resulted in normalization of both the platelet count and PAIgG level. It is suggested that plasmapheres is removes antiplatelet antibody or immune complexes which may be of etiologicalimportance in this illness.

Safety and Efficacy of Mycophenolate Mofetil in Relapsing Acquired Thrombotic Thrombocytopenic Purpura: Could It Prevent Further Relapse?.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3993-3993 ◽  
Author(s):  
Sameer A. Tulpule ◽  
Yvonne A. Francis ◽  
Deepti Radia ◽  
Claire N. Harrison ◽  
Beverely J. Hunt
Keyword(s):  
Platelet Count ◽  
Mycophenolate Mofetil ◽  
Plasma Exchange ◽  
Thrombotic Thrombocytopenic Purpura ◽  
Maximum Dose ◽  
Case Series ◽  
Initial Presentation ◽  
Thrombocytopenic Purpura ◽  
Methyl Prednisolone ◽  
Threatening Condition

Abstract Introduction: Thrombotic thrombocytopenic purpura (TTP) is a life threatening condition requiring rapid diagnosis and treatment. Plasma exchange (PEX) is the mainstay of treatment. Various forms of immunosuppression (IS) have been used which include steroids, cyclosporine, cyclophosphamide, vincristine and rituximab. The percentage of patients relapsing is unclear. There is a suggestion that up to half of the patients with severe acquired deficiency of von Willebrand factor -cleaving protease (vWF-CP) activity relapse within a year. There are no reports of the use of mycophenolate mofetil (MMF) in acquired TTP. We describe three patients with acquired TTP, treated with MMF at relapse, with the intention to prevent further relapse. Methods: The 3 patients presented with acute acquired TTP. They all had at least 3 of the clinical pentad of fever, microangiopathic haemolytic anaemia, thrombocytopenia, neurological and renal impairment plus a vWF-CP level of &lt; 2% at initial presentation. All of them underwent PEX until remission (platelet count of &gt;150 x 109/L for at least 2 consecutive days with resolution of neurological and renal signs). MMF was introduced at remission after relapse at a dose of 500mg BD, post PEX, increasing upto a maximum dose of 750 mg BD. MMF was introduced at 4th relapse for patient A, 2nd relapse for patient B and 1st relapse for patient C. Results: All 3 patients were females. The ages at presentation were 63, 72 and 46 years. At presentation, the haemoglobin was 6.0, 8.7 and 6.7 g/dL and platelet count was 19, 36 and 21 x 109 /L respectively. Patient A relapsed eight times at day (d) 9, d20, d53, d89, d198, d209, d221 and d231. She was treated with PEX in conjunction with steroids and vincristine, cyclosporine, cyclophosphamide and rituximab for the first 3 relapses respectively. During the third relapse the patient’s condition deteriorated and she became unconcious requiring ventilation. MRI brain showed multiple small foci consistent with vascular disease. She recovered, but relapsed again despite cyclophosphamide and rituximab. After the 4th relapse on d102, MMF was started reaching a maximum dose of 750mg BD. She had regular full blood counts checked. At d187 she was found to be neutropenic and the MMF was stopped. She relapsed in 11 days and was recommenced on MMF at 500mg bd after PEX. MMF was continued at the dose of 750mg BD after the 7th and 8th relapse. Despite full dose MMF, she relapsed and was treated with PEX and a further course of rituximab was given at the 8th relapse. Patient B had received 500mg of methyl prednisolone on ITU with PEX at initial presentation. MMF (500mg BD) was commenced at remission after second relapse (d23) after undergoing plasma exchange. Patient C was commenced on MMF (500mg BD) after first relapse (d36). All 3 are in remission and continue on MMF at a follow up of 12, 2 and 4 months respectively since last relapse. MMF was tolerated very well except for transient neutropenia (patient A) and transient diarrhoea (patient C). Conclusion: MMF appears to be safe in patients with relapsed TTP who received multiple lines of treatment. Due to the small size of this case series it is unclear whether MMF is efficacious in reducing the risk of relapse in TTP; a formal longer study may be warranted.

Ectopic Expression of ADAMTS13 in Platelets As a Novel Therapeutic Approach for Arterial Thrombosis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 491-491
Author(s):  
Brandy Pickens ◽  
Sheng-Yu Jin ◽  
Dengju Li ◽  
X. Long Zheng
Keyword(s):  
Transgenic Mice ◽  
Proteolytic Activity ◽  
Targeted Delivery ◽  
Arterial Thrombosis ◽  
Thrombus Formation ◽  
Clotting Factor ◽  
Platelet Glycoprotein ◽  
Wild Type ◽  
F1 Hybrid ◽  
Novel Therapeutic

Abstract Abstract 491 Megakaryocytes and platelets have been shown to produce ADAMTS13 and its only known substrate, von Willebrand factor (VWF). However, the role of platelet expression of ADMTS13 in modulation of thrombus formation is not known. Previous studies have shown that platelet-targeted delivery of clotting factor VIII corrects bleeding phenotype in hemophilia A mice despite of inhibitors. These results suggest that platelet-delivery of ADAMTS13 may also be efficacious for anti-arterial thrombosis and perhaps for treatment of acquired idiopathic thrombotic thrombocytopenic purpura (TTP) with inhibitors. In the present study, transgenic mice (JAX B6SJL/F1 hybrid) carrying a human full-length ADAMTS13 gene under a platelet glycoprotein 1b alpha promoter were generated. The mice were crossed with Adamts13−/− and TTP-sensitive mice (CAST/Ei) for 4 generations. Plasma and platelet ADAMTS13 protein and proteolytic activity were determined. By Western blotting and the cleavage of a fluorescein-labeled VWF73 substrate, we were able to show that human ADAMTS13 protein (∼195 kDa) and activity were present in the platelet lysate of transgenic (A13-PltTG) mice, but not in adamts13−/− mice or wild-type mice. No proteolytic activity was detected in plasma of the transgenic mice. The platelet ADAMTS13 protein was releasable upon stimulation with various concentrations of thrombin (0.1–0.5 U/ml) and collagen (2.5–10 μg/ml). The released ADAMTS13 and VWF (as a positive control) were primarily associated with platelet membrane, demonstrated by surface biotinylation. However, a small fraction of the released ADAMTS13 and VWF proteins were detected in the releasate after stimulation. Moreover, the A13-PltTG mice exhibited systemic anti-thrombotic activity, which attenuated the rate of thrombus formation in the mesenteric arterioles induced by a topical application of 10% ferric chloride. The rate of arterial thrombus formation in the transgenic mice was significantly lower than that in Adamts13−/−mice and wild-type mice in the same genetic background. We conclude that we have generated transgenic mice overexpressing human ADAMTS13 metalloprotease in platelets. The platelet expressed ADAMTS13 is releasable upon stimulation by agonists. The platelet derived ADAMTS13 is biologically functional in cleaving VWF in vitro and in vivo, which attenuate systemic arterial thrombosis after oxidative injury. Our ongoing effort is to determine the efficacy of platelet delivered ADAMTS13 as a potential novel therapeutic for acquired TTP patients with inhibitors. Disclosures: No relevant conflicts of interest to declare.

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