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1.  Thrombin-Targeted Liposomes Establish A Sustained Localized Anticlotting Barrier Against Acute Thrombosis 
Molecular pharmaceutics  2013;10(11):4168-4175.
The goal of the present work was to design and test an acute-use nanoparticle-based antithrombotic agent that exhibits sustained local inhibition of thrombin without requiring a systemic anticoagulant effect to function against acute arterial thrombosis. To demonstrate proof of concept, we functionalized the surface of liposomes with multiple copies of the direct thrombin inhibitor, D-phenylalanyl-L-prolyl-L-arginyl-chloromethyl ketone (PPACK), which exhibits high affinity for thrombin as a free agent, but manifests too rapid clearance in vivo to be effective alone. The PPACK-Liposomes were formulated as single unilamellar vesicles, with a diameter of 170.78 ± 10.59 nm and a near neutral charge. In vitro models confirmed the inhibitory activity of PPACK-Liposomes, demonstrating a KI′ of 172.6 nM. In experimental clots in vitro, treatment of formed clots completely abrogated any further clotting upon exposure to human plasma. The liposomes were evaluated in vivo in a model of photochemical-induced carotid artery injury, resulting in significantly prolonged arterial occlusion time over that of controls (69.06 ± 5.65 min for saline treatment, N=6, 71.33 ± 9.46 min for free PPACK treated 85.75 ± 18.24 min for precursor liposomes; N=4, 139.75 ± 20.46 min for PPACK-Liposomes; P = 0.0049, N=6). Systemic anticoagulant profiles revealed a rapid return to control levels within 50 minutes, while still maintaining antithrombin activity at the injury site. The establishment of a potent and long-acting anticoagulant surface over a newly forming clot with the use of thrombin targeted nanoparticles that do not require systemic anticoagulation to be effective offers an alternative site-targeted approach to the management of acute thrombosis.
PMCID: PMC3946534  PMID: 24063304
Drug Delivery; Liposomes; Nanoparticles; Anticoagulants; Thrombosis
2.  A Structural Explanation for the Antithrombotic Activity of ARC1172, a DNA Aptamer that Binds von Willebrand Factor Domain A1 
Structure (London, England : 1993)  2009;17(11):10.1016/j.str.2009.09.011.
ARC1172 is a 41-mer DNA aptamer selected to bind the A1 domain of von Willebrand factor (VWF). A derivative of ARC1172 with modifications to increase intravascular survival inhibits carotid artery thrombosis in a Cynomolgus macaque model and inhibits VWF-dependent platelet aggregation in humans, suggesting that such aptamers may be useful to prevent or treat thrombosis. In the crystal structure of a VWF A1-ARC1172 complex, the aptamer adopts a three-stem structure of mainly B-form DNA with three noncanonical base pairs and 9 unpaired residues, 6 of which are stabilized by base-base or base-deoxyribose stacking interactions. The aptamer-protein interface is characterized by cation-π interactions involving Arg, Lys and Gln residues, often stabilized by H-bonds with adjacent bases. The ARC1172 binding site on the A1 domain overlaps with that of botrocetin and clashes with glycoprotein Ibα binding at an adjacent site, which accounts for the antithrombotic activity of ARC1172 and related aptamers.
PMCID: PMC3845234  PMID: 19913482
3.  Accelerated clearance alone explains ultralarge multimers in VWD Vicenza 
Journal of thrombosis and haemostasis : JTH  2010;8(6):10.1111/j.1538-7836.2010.03753.x.
von Willebrand disease Vicenza is characterized by low plasma VWF levels, the presence of ultra-large (UL) VWF multimers and less prominent satellite bands on multimer gels, and the heterozygous amino acid substitution R1205H in the VWF gene. The pathogenesis of VWD Vicenza has been elusive. Accelerated clearance is implicated as a cause of low VWF level.
We addressed the question, whether the presence of ultra-large multimers is a cause, or a result of accelerated VWF clearance, or whether it is an unrelated phenomenon.
We studied the detailed phenotype of three Hungarian patients with VWD Vicenza, expressed the mutant VWF-R1205H in 293T cells, and developed a mathematical model to simulate VWF synthesis and catabolism.
We found that the half life of VWF after DDAVP was approximately one tenth of that after the administration of Haemate P, a source of exogenous wild type VWF (0.81±0.2 vs. 7.25±2.38 hours). An analysis of recombinant mutant VWF-R1205H showed that the biosynthesis and multimer structure of WT and mutant VWF were indistinguishable. A mathematical model of the complex interplay of VWF synthesis, clearance and cleavage showed that decreasing VWF half life to one tenth of normal reproduced all features of VWD Vicenza including low VWF level, ultra-large multimers and a decrease of satellite band intensity.
We conclude that accelerated clearance alone may explain all features of VWD Vicenza.
PMCID: PMC3863617  PMID: 20088930
von Willebrand disease; von Willebrand factor; Clearance
4.  Two Mechanistic Pathways for Thienopyridine-Associated Thrombotic Thrombocytopenic Purpura 
We sought to describe clinical and laboratory findings for a large cohort of patients with thienopyridine-associated thrombotic thrombocytopenic purpura (TTP).
The thienopyridine derivatives, ticlopidine and clopidogrel, are the 2 most common drugs associated with TTP in databases maintained by the U.S. Food and Drug Administration (FDA).
Clinical reports of TTP associated with clopidogrel and ticlopidine were identified from medical records, published case reports, and FDA case reports (n = 128). Duration of thienopyridine exposure, clinical and laboratory findings, and survival were recorded. ADAMTS13 activity (n = 39) and inhibitor (n = 30) were measured for a subset of individuals.
Compared with clopidogrel-associated TTP cases (n = 35), ticlopidine-associated TTP cases (n = 93) were more likely to have received more than 2 weeks of drug (90% vs. 26%), to be severely thrombocytopenic (84% vs. 60%), and to have normal renal function (72% vs. 45%) (p < 0.01 for each). Compared with TTP patients with ADAMTS13 activity >15% (n = 13), TTP patients with severely deficient ADAMTS13 activity (n = 26) were more likely to have received ticlopidine (92.3% vs. 46.2%, p < 0.003). Among patients who developed TTP >2 weeks after thienopyridine, therapeutic plasma exchange (TPE) increased likelihood of survival (84% vs. 38%, p < 0.05). Among patients who developed TTP within 2 weeks of starting thienopyridines, survival was 77% with TPE and 78% without.
Thrombotic thrombocytopenic purpura is a rare complication of thienopyridine treatment. This drug toxicity appears to occur by 2 different mechanistic pathways, characterized primarily by time of onset before versus after 2 weeks of thienopyridine administration. If TTP occurs after 2 weeks of ticlopidine or clopidogrel therapy, therapeutic plasma exchange must be promptly instituted to enhance likelihood of survival.
PMCID: PMC3167088  PMID: 17868804
5.  Identification of amino acid residues responsible for von Willebrand factor binding to sulfatide by charged-to-alanine-scanning mutagenesis 
von Willebrand factor (VWF) performs its hemostatic functions through binding to various proteins. The A1 domain of VWF contains binding sites of not only physiologically important ligands, but also exogenous modulators that induce VWF-platelet aggregation. Sulfatides, 3-sulfated galactosyl ceramides, that are expressed on oligodendrocytes, renal tubular cells, certain tumor cells and platelets, have been suggested to interact with VWF under some pathological conditions. The binding of VWF to sulfatide requires the A1 domain, but its binding sites have not been precisely identified. Here, we report that alanine mutations at Arg1392, Arg1395, Arg1399 and Lys1423 led to decreased VWF–sulfatide binding. These sites have been reported to be the binding sites for platelet membrane glycoprotein (GP) Ib and/or snake venom botrocetin, and, interestingly, are identical to the monoclonal antibody (mAb) NMC4 epitope previously reported to inhibit the VWF-GPIb interaction. We observed that NMC4 also inhibited VWF interaction with sulfatides in a dose-dependent manner. Thus, we conclude that VWF binding sites of sulfatide overlap those of platelet GPIb and botrocetin.
PMCID: PMC2668596  PMID: 18369690
von Willebrand factor; A1 domain; Sulfatide; Alanine scanning mutagenesis
6.  Pathogenesis of Thrombotic Microangiopathies 
Annual review of pathology  2008;3:249-277.
Profound thrombocytopenia and microangiopathic hemolytic anemia characterize thrombotic microangiopathy, which includes two major disorders: thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS). TTP has at least three types: congenital or familial, idiopathic, and nonidiopathic. The congenital and idiopathic TTP syndromes are caused primarily by deficiency of ADAMTS13, owing to mutations in the ADAMTS13 gene or autoantibodies that inhibit ADAMTS13 activity. HUS is similar to TTP, but is associated with acute renal failure. Diarrhea-associated HUS accounts for more than 90% of cases and is usually caused by infection with Shiga-toxin-producing Escherichia coli (O157:H7). Diarrhea-negative HUS is associated with complement dysregulation in up to 50% of cases, caused by mutations in complement factor H, membrane cofactor protein, factor I or factor B, or by autoanti-bodies against factor H. The incomplete penetrance of mutations in either ADAMTS13 or complement regulatory genes suggests that precipitating events or triggers may be required to cause thrombotic microangiopathy in many patients.
PMCID: PMC2582586  PMID: 18215115
thrombotic thrombocytopenic purpura; hemolytic uremic syndrome; von Willebrand factor-cleaving metalloprotease; ADAMTS13; complement dysregulation

Results 1-6 (6)