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.

ADAMTS13 gene deletion aggravates ischemic brain damage: a possible neuroprotective role of ADAMTS13 by ameliorating postischemic hypoperfusion

Blood ◽  
2010 ◽  
Vol 115 (8) ◽  
pp. 1650-1653 ◽  
Author(s):  
Masayuki Fujioka ◽  
Kazuhide Hayakawa ◽  
Kenichi Mishima ◽  
Ai Kunizawa ◽  
Keiichi Irie ◽  
...  
Keyword(s):  
Brain Damage ◽  
Gene Deletion ◽  
Ischemia Reperfusion Injury ◽  
Thrombus Formation ◽  
Infarct Volume ◽  
Ischemia Reperfusion ◽  
Platelet Glycoprotein ◽  
Wild Type ◽  
Ischemic Brain Damage ◽  
Ischemic Brain

Abstract Reperfusion after brain ischemia causes thrombus formation and microcirculatory disturbances, which are dependent on the platelet glycoprotein Ib–von Willebrand factor (VWF) axis. Because ADAMTS13 cleaves VWF and limits platelet-dependent thrombus growth, ADAMTS13 may ameliorate ischemic brain damage in acute stroke. We investigated the effects of ADAMTS13 on ischemia-reperfusion injury using a 30-minute middle cerebral artery occlusion model in Adamts13−/− and wild-type mice. After reperfusion for 0.5 hours, the regional cerebral blood flow in the ischemic cortex was decreased markedly in Adamts13−/− mice compared with wild-type mice (P < .05), which also resulted in a larger infarct volume after 24 hours for Adamts13−/− compared with wild-type mice (P < .01). Thus, Adamts13 gene deletion aggravated ischemic brain damage, suggesting that ADAMTS13 may protect the brain from ischemia by regulating VWF-platelet interactions after reperfusion. These results indicate that ADAMTS13 may be a useful therapeutic agent for stroke.

Functional Genomics in Zebrafish Permits Rapid Characterization of Novel Platelet Membrane Proteins

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2860-2860
Author(s):  
Isabelle I Salles ◽  
Marie N O’Connor ◽  
Nicholas A. Watkins ◽  
Adam Walker ◽  
Stephen F Garner ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Arterial Thrombosis ◽  
Thrombus Formation ◽  
Platelet Membrane ◽  
Platelet Glycoprotein ◽  
Functional Screening ◽  
Phenotypic Analysis ◽  
Spontaneous Bleeding ◽  
Platelet Proteins

Abstract In this study we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell the megakaryocyte, together with nucleated blood cell elements, endothelial cells and erythroblasts, identified novel platelet membrane proteins with hitherto unknown role in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)-based knockdown of five of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet Glycoprotein (GP) IIb and the coagulation protein Factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of four of the five novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis.

Specific Pharmacological Targeting of the Syk Kinase Activity in Platelets: A Novel, Safe Anti-Thrombotic Strategy

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 409-409 ◽  
Author(s):  
Suzanne Delaney ◽  
Uma Sinha ◽  
Nisha Nanda ◽  
Yibing Yan ◽  
Anjali Pandey ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Arterial Thrombosis ◽  
Kinase Activity ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Wild Type ◽  
Primary Hemostasis ◽  
Syk Kinase ◽  
Perfusion Chamber

Abstract Studies of the Syk −/− mouse have implicated spleen tyrosine kinase (Syk), a signaling protein with both kinase and scaffolding activities, in platelet signaling following engagement of GPVI and αIIbβ3 by collagen and fibrinogen, respectively. The present study was designed to determine whether specific inhibition of the kinase activity of Syk, without targeting the Syk scaffolding function, affected in vivo arterial thrombosis. In preliminary experiments, blood from wild-type and Syk−/− mice was perfused through collagen-coated capillaries under arterial shear rates to study ex vivo thrombosis. While blood from wild-type mice formed robust thrombi (37±4.7 μm3/μm2), none was observed in Syk−/− mice. Thrombi intermediate in size (16±3.9 μm3/μm2) developed in Syk+/− mice. To achieve specific pharmacological targeting of the kinase activity of Syk, P142-76, a potent (IC50 = 4 nM) and selective Syk kinase inhibitor was utilized. P142-76 was screened against a broad panel of 139 purified kinases at 50 nM. While Syk kinase was inhibited by 92%, all other kinases retained more than 70% of their activity. In washed human platelets, P142-76 inhibited convulxin (CVX)-induced phosphorylation of LAT (linker for activation of T-cells; IC50 = 111 nM) and intracellular calcium increases (IC50 = 31 nM). The GPVI/Syk-specificity of P142-76 activity was confirmed by its inability to inhibit intracellular calcium increases induced by the PAR1 thrombin receptor agonist TRAP. P142-76 also inhibited CVX-induced aggregation of both human washed platelets (IC50 = 87 nM) and platelet-rich plasma (IC50 = 2.5 μM). Considering the controversial data in respect to the participation of GPVI in arterial thrombosis in murine models, the dependence of arterial thrombosis on Syk function was studied in vivo in pigs. Cross-species activity of P142-76 was confirmed in vitro (CVX-induced PRP aggregation IC50= 350 nM; 5 μM P142-76 completely inhibited thrombosis triggered by collagen in the perfusion chamber assay). At a plasma concentration which abolished ex vivo CVX-induced but not ADP-induced pig platelet aggregation, P142-76 significantly inhibited the deposition of [111In]-labeled platelets in a carotid artery crush swine thrombosis model, without compromising primary hemostasis. % aggregation Swine (n=3) Platelet Deposition % inhibition Plasma Conc (ng/ml) Bleed Time (min) Activated Clotting Time (sec) ADP (20 μM) CVX (250 ng/ml) Control Artery 0 0 3±0.9 133±22 100 100 Treated Artery 76±6.5 1343±304 3.5±0.3 130±13 100 0 To clarify further the contribution of the kinase activity of Syk to arterial thrombosis, effects of P142-76 on human blood were evaluated in real time in the collagen-coated perfusion chamber. Low concentrations of P142-76 (0.3 μM) affected thrombus stability, while increasing concentrations (1–5 μM) delayed and then completely inhibited thrombus formation. Furthermore, P142-76 destabilized pre-formed thrombi, indicating a critical role for Syk in conferring strength to platelet-platelet interactions, i.e. αIIbβ3-mediated cohesion. Our data indicate that the kinase activity of Syk acts in arterial thrombosis through at least two distinct mechanisms. First, Syk kinase confers stability to platelet-platelet interactions downstream of αIIbβ3. Second, it initiates thrombus formation on collagen surfaces. This dual activity of the kinase activity of Syk makes it a preferred target for inhibition of arterial thrombosis, as it does not compromise primary hemostasis.

The Role of the VWF A1 and A2 Domains in Murine Models of TTP and Thrombosis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3060-3060
Author(s):  
Jennifer Barr ◽  
Justin Barr ◽  
David Motto
Keyword(s):  
Thrombus Formation ◽  
Platelet Glycoprotein ◽  
Cysteine Residues ◽  
Wild Type ◽  
Domain Sequence ◽  
Initial Adhesion ◽  
A1 Domain ◽  
A2 Domain

Abstract Abstract 3060 Poster Board II-1036 von Willebrand Factor (VWF) is a large multimeric plasma glycoprotein synthesized in endothelial cells and megakaryocytes. In humans and mice, VWF dysfunction is associated both with defects in hemostasis, and with the systemic blood clotting disease thrombotic thrombocytopenic purpura (TTP). The initial adhesion of platelets to sites of vascular injury in large part involves binding of the VWF A1 domain to the platelet glycoprotein receptor GPIb alpha. This VWF A1-GPIb alpha interaction, along with deficiency of the ADAMTS13 plasma metalloprotease, is thought to be required for the pathogenesis of TTP. Deficiency of ADAMTS13 results in the failure to cleave the Y1605-M1606 sissile bond within the A2 domain of VWF. The structure of VWF is strongly influenced by its high content of cysteine residues, all of which are involved in inter-or intra-chain disulfide bonds. The location of these cysteine residues within the A domains leads to the formation of disulfide loops within the A1 (Cys 1272-1458) and A3 (Cys 1686-1872) domains, but not within the A2 domain. The lack of a disulfide loop allows the A2 domain to assume a “flexible” conformation that is thought to “open” in response to fluid shear stress, exposing the Y1605-M1606 bond to cleavage by ADAMTS13. To investigate specifically the role of the VWF A1-Gp1b alpha interaction in the context of otherwise functional VWF in vivo, we generated a chimeric murine VWF expression construct in which the murine A1 domain sequence is replaced with the corresponding sequence from human VWF (the human VWF A1 domain is known to not interact appreciably with murine GPIb alpha). Additionally, we engineered a VWF construct in which paired cysteine residues analogous to those in the A1 and A3 domains were introduced into the A2 domain sequence, with the goal being to “lock” the A2 domain closed and prevent cleavage by ADAMTS13. Hydrodynamic tail vein injection of both the VWF-hA1 and the VWF-A2 lock constructs into VWF-deficient mice resulted in plasma VWF levels up to 20-fold higher than observed in wild-type mice, dependent on the amount of plasmid injected. Importantly, the degree of VWF multimerization appeared nearly identical both to that observed in wild-type mice, and to mice injected with wild-type murine VWF, and expression persisted for approximately 30 days. Functionally, unlike WT murine VWF, expression of VWF-hA1 failed to restore thrombus formation in a ferric chloride-induced injury model, demonstrating the crucial importance of the VWF A1-GP1b apha interaction in thrombus formation. Currently we are investigating whether expression of VWF-hA1 can support disease pathogenesis in a mouse model of TTP. Similarly, we are determining whether expression of VWF-A2 lock leads to development of TTP, even in the presence of ADAMTS13. The ultimate goal of these studies is to completely “humanize” the VWF A1-GP1b alpha interaction in mice by replacing the murine GP1b alpha sequence with that from humans. These resulting animals will be used to further investigate the role of the VWF A1 domain-GPIb alpha interaction in vivo, and should prove useful for identifying compounds to effectively inhibit this interaction in humans. In addition, the expression of a VWF construct that is unable to be cleaved by ADAMTS13 should help to elucidate the role of VWF cleavage in TTP pathogenesis. Disclosures No relevant conflicts of interest to declare.

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.

Multiple Carboxyl-Terminal Domains of ADAMTS13 Are Required for Systemic Anti-Arterial Thrombosis In Vivo

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 24-24 ◽  
Author(s):  
Juan (Jenny) Xiao ◽  
Sheng-Yu Jin ◽  
X. Long Zheng
Keyword(s):  
Ferric Chloride ◽  
Arterial Thrombosis ◽  
Thrombus Formation ◽  
Arterial Occlusion ◽  
Final Concentration ◽  
Wild Type ◽  
The Times ◽  
Carotid Arterial ◽  
Terminal Domains

Abstract Abstract 24 A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats (ADAMTS)-13 cleaves newly released ultra large (UL) von Willebrand factor (VWF), thereby inhibiting excessive platelet aggregation and thrombus formation. Inability to cleave ULVWF due to deficiency of plasma ADAMTS13 activity may result in thrombotic thrombocytopenic purpura (TTP), a potentially fatal illness, and other arterial thrombotic diseases (i.e. myocardial and cerebral infarctions). However, little is known about the structural components of ADAMTS13 required for systemic anti-arterial thromboses in vivo. In this study, we determined the biological effect of ADAMTS13 and variants on arterial thromboses in a murine model using two different assays. First, using a ferric chloride-induced carotid arterial occlusion assay, we demonstrated that the times to a complete occlusion (TCO) of carotid artery after topical application of 10% ferric chloride (soaked in a filter paper 1×2 min, for 2 min) in wild-type (C57BL/6) mice and Adamts13-/- mice were 10.0 ± 1.0 min (mean ± SEM) (n=9) and 5.3 ± 0.4 min (n=10), respectively. The difference was statistically highly significant (p<0.0001). An infusion of 10 nM of recombinant human full-length ADATMS13 (FL), a variant truncated after the 8th TSP1 repeat (T8) and after the spacer domain (S) into the Adamts13-/- mice restored the TCO to 12.7 ± 1.7 min (n=12), 8.0 ± 1.8 (n=7), and 22.0 ± 2.1 min (n=12), respectively. These results suggest that the N-terminal fragment of ADAMTS13 up to the spacer domain is sufficient for protection against ferric chloride induced arterial thrombosis. Moreover, an infusion of an ADAMTS13 mutant lacking 6 amino acid residues between Arg659 and Glu664 (d6a) into Adamts13-/-mice did not restore the TCO (5.9 ± 0.6 min, n=11), suggesting the critical role of the spacer domain in anti-arterial thrombosis in vivo. Paradoxically, however, an infusion of a recombinant C-terminal fragment of ADAMTS13 consisting of the TSP1 5–8 repeats and CUB domains (T5C) at the final concentration of 50 nM (10x endogenous murine plasma ADAMTS13 concentration) into wild-type mice significantly shortened the TCO (5.9 ± 1.9 min) (n=11), similar to that in the Adamts13-/- mice receiving injection of PBS alone (p=0.440). These results indicate that the middle and distal C-terminal domains of ADAMTS13 in the context of the whole molecule may also participate in substrate recognition and are required for anti-arterial thrombotic function in vivo. Second, the results from intravital microscopy by visualizing a real-time thrombus formation in the mesenteric arterioles nearly mirrored the data obtained by the carotid arterial occlusion assay. In these experiments, the times to an initial thrombus formation (Ti) (defined as the time to form a thrombus >30 μ m) and the times to a complete occlusion of blood vessel (Tc) were determined. We showed that the Ti and Tc in wild-type C57BL6 mice were 8.8 ± 0.6 min (mean ± SEM) and 13.7 ± 1.0 min (n=12), respectively. The Ti and Tc in the Adamts13-/- mice (same genetic background) were 5.2 ± 0.6 min (n=15) and 9.6 ± 0.8 min (n=15), respectively. The differences in both Ti and Tc between wild-type mice and Adamts13-/- mice were statistically highly significant (p<0.001). An infusion of recombinant human FL, T8, and S, but not d6a at a final concentration of 10 nM significantly prolonged the Ti (FL: 9.7 ± 0.9 min, n=10; T8: 10.1 ± 1.5 min, n=10; S: 11.5 ± 1.5 min, n=10; d6a: 5.3 ± 0.57 min, n=11) and the Tc (FL: 15.3 ± 1.4 min; T8: 21.9 ± 2.2 min; S: 16.2 ± 1.6 min; d6a:10.0 ± 1.1 min). The differences in both Ti and Tc between the control (buffer injected) and experimental groups were all statistically highly significant. We conclude that multiple C-terminal domains of ADAMTS13 are required for systemic anti-arterial/arteriolar thrombosis under (patho) physiological conditions. Our findings may shed more light on pathogenesis of TTP and provide molecular basis for a rational design of novel therapies for TTP and perhaps other arterial thrombotic disorders because of dysfunction of VWF/ADAMTS13 axis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Platelets Loaded with Recombinant ADAMTS13 Are Efficacious in Inhibiting Arterial Thrombosis Under Flow: A Potential Novel Therapeutic for Acquired TTP

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 763-763
Author(s):  
Mohammad Abdelgawwad ◽  
Liang Zheng ◽  
X. Long Zheng
Keyword(s):  
Ferric Chloride ◽  
Arterial Thrombosis ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Dependent Manner ◽  
Novel Therapy ◽  
Concentration Dependent Manner ◽  
Novel Therapeutic

Abstract Background: Thrombotic thrombocytopenic purpura (TTP), a potentially life-threatening blood clotting disorder, is resulted from severe deficiency of ADAMTS13 activity. Plasma exchange is the only effective initial therapy for acquired TTP, which replenishes ADAMTS13 while removing autoantibodies. However, the in-hospital mortality rate is still high, suggesting that another more effective therapy is urgently needed. Objectives: The present study aims to develop a novel therapy using platelets as carriers to deliver recombinant ADAMTS13 (rADAMTS13) to the sites of vascular injury, where platelets are activated and de-granulated to release rADAMTS13 from the storage granules. Our specific aims are: 1) determine the in vitro uptake of rADAMTS13 by platelets; 2) determine the efficacy of platelet-delivered rADAMTS13 in anti-arterial thrombosis under flow and in vivo. Methods: Freshly isolated human platelets from whole blood were incubated with an increasing concentration of rADAMTS13 (0, 5, 20, 50, and 100 µg/ml) at 25°C and 37°C for hours. Platelets were then washed and lysed with 1% Triton X-100 to assess the rADAMTS13 activity via a FRETS-VWF73 assay. Western blotting and confocal microscopy determined the amounts of rADAMTS13 protein and localization in platelets, respectively. The anti-thrombotic activity of the released rADAMTS13 from activated platelets was determined by a microfluidic system (BioFlux) and by a ferric chloride induced thrombus formation in mesenteric arterioles/venules in Adamts13-/- mice. Results: Human washed platelets were able to uptake rADAMTS13 in a temperature- and concentration-dependent manner. The rADAMTS13 protein packed inside human platelets remained intact and active towards the FRET-VWF73 substrate. Confocal microscopic analysis demonstrated its localization of uptaken rADAMTS13 in the alpha granules, partially overlapped with VWF. The rADAMTS13 released from activated human platelets under shear (100 dyne/cm2) was capable of cleaving VWF and inhibiting platelet agglutination and aggregation on a collagen-coated surface in a concentration-dependent manner. Transfusion of platelets expressing rADAMTS13 into Adamts13-/- mice dramatically reduced the rate of thrombus formation in the mesenteric arterioles/venules after ferric chloride injury. Conclusions: Our results demonstrate that human platelets can be reengineered in vitro to contain sufficient amounts of rADAMTS13 in the alpha granules. The rADAMTS13 inside platelets is releasable and proteolytically functional under shear stress or in vivo under flow. Our findings suggest that the transfusion of platelets loaded with rADAMTS13 may represent a novel therapeutic strategy for acquired TTP with inhibitors. Disclosures Zheng: Alexion: Speakers Bureau; Ablynx: Consultancy.

scholarly journals Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins

Blood ◽  
2009 ◽  
Vol 113 (19) ◽  
pp. 4754-4762 ◽  
Author(s):  
Marie N. O'Connor ◽  
Isabelle I. Salles ◽  
Ana Cvejic ◽  
Nicholas A. Watkins ◽  
Adam Walker ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Arterial Thrombosis ◽  
Thrombus Formation ◽  
Platelet Membrane ◽  
Platelet Glycoprotein ◽  
Functional Screening ◽  
Phenotypic Analysis ◽  
Spontaneous Bleeding ◽  
Platelet Proteins

Abstract In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)–based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis.

Differential Role of Components of the Fibrinolytic System in the Formation and Removal of Thrombus Induced by Endothelial Injury

1999 ◽  
Vol 81 (04) ◽  
pp. 601-604 ◽  
Author(s):  
Hiroyuki Matsuno ◽  
Osamu Kozawa ◽  
Masayuki Niwa ◽  
Shigeru Ueshima ◽  
Osamu Matsuo ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Thrombus Formation ◽  
Endothelial Injury ◽  
Fibrinolytic System ◽  
Tissue Type ◽  
Clot Lysis ◽  
Wild Type ◽  
Type Plasminogen Activator ◽  
Pai 1

SummaryThe role of fibrinolytic system components in thrombus formation and removal in vivo was investigated in groups of six mice deficient in urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), or plasminogen activator inhibitor-1 (PAI-1) (u-PA-/-, t-PA-/- or PAI-1-/-, respectively) or of their wild type controls (u-PA+/+, t-PA+/+ or PAI-1+/+). Thrombus was induced in the murine carotid artery by endothelial injury using the photochemical reaction between rose bengal and green light (540 nm). Blood flow was continuously monitored for 90 min on day 0 and for 20 min on days 1, 2 and 3. The times to occlusion after the initiation of endothelial injury in u-PA+/+, t-PA+/+ or PAI-1+/+ mice were 9.4 ± 1.3, 9.8 ± 1.1 or 9.7 ± 1.6 min, respectively. u-PA-/- and t-PA-/- mice were indistinguishable from controls, whereas that of PAI-1-/- mice were significantly prolonged (18.4 ± 3.7 min). Occlusion persisted for the initial 90 min observation period in 10 of 18 wild type mice and was followed by cyclic reflow and reocclusion in the remaining 8 mice. At day 1, persistent occlusion was observed in 1 wild type mouse, 8 mice had cyclic reflow and reocclusion and 9 mice had persistent reflow. At day 2, all injured arteries had persistent reflow. Persistent occlusion for 90 min on day 0 was observed in 3 u-PA-/-, in all t-PA-/- mice at day 1 and in 2 of the t-PA-/-mice at day 2 (p <0.01 versus wild type mice). Persistent patency was observed in all PAI-1-/- mice at day 1 and in 5 of the 6 u-PA-/- mice at day 2 (both p <0.05 versus wild type mice). In conclusion, t-PA increases the rate of clot lysis after endothelial injury, PAI-1 reduces the time to occlusion and delays clot lysis, whereas u-PA has little effect on thrombus formation and spontaneous lysis.

Platelet Activation and Aggregation Induced by Recombinant von Willebrand Factors Reproducing Four Type 2B von Willebrand Disease Missense Mutations

1998 ◽  
Vol 79 (01) ◽  
pp. 211-216 ◽  
Author(s):  
Lysiane Hilbert ◽  
Claudine Mazurier ◽  
Christophe de Romeuf
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Von Willebrand Disease ◽  
Platelet Glycoprotein ◽  
Missense Mutations ◽  
Inhibit Platelet Aggregation ◽  
Wild Type ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryType 2B of von Willebrand disease (vWD) refers to qualitative variants with increased affinity of von Willebrand factor (vWF) for platelet glycoprotein Ib (GPIb). All the mutations responsible for type 2B vWD have been located in the A1 domain of vWF. In this study, various recombinant von Willebrand factors (rvWF) reproducing four type 2B vWD missense mutations were compared to wild-type rvWF (WT-rvWF) for their spontaneous binding to platelets and their capacity to induce platelet activation and aggregation. Our data show that the multimeric pattern of each mutated rvWF is similar to that of WT-rvWF but the extent of spontaneous binding and the capacity to induce platelet activation and aggregation are more important for the R543Q and V553M mutations than for the L697V and A698V mutations. Both the binding of mutated rvWFs to platelets and platelet aggregation induced by type 2B rvWFs are inhibited by monoclonal anti-GPIb and anti-vWF antibodies, inhibitors of vWF binding to platelets in the presence of ristocetin, as well as by aurin tricarboxylic acid. On the other hand, EDTA and a monoclonal antibody directed against GPIIb/IIIa only inhibit platelet aggregation. Furthermore, the incubation of type 2B rvWFs with platelets, under stirring conditions, results in the decrease in high molecular weight vWF multimers in solution, the extent of which appears correlated with that of plasma vWF from type 2B vWD patients harboring the corresponding missense mutation. This study supports that the binding of different mutated type 2B vWFs onto platelet GPIb induces various degrees of platelet activation and aggregation and thus suggests that the phenotypic heterogeneity of type 2B vWD may be related to the nature and/or location of the causative point mutation.

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