The metalloprotease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin motif) converts the hyperreactive unusually large (UL) forms of von Willebrand factor (VWF) that are newly released ...from endothelial cells into less active plasma forms by cleaving a peptide bond in the VWF A2 domain. Familial or acquired deficiency of this metalloprotease is associated with thrombotic thrombocytopenic purpura (TTP). ADAMTS13 belongs to the ADAMTS metalloprotease family, but, unlike other members, it also contains 2 C-terminal CUB domains (complement component Clr/Cls, Uegf, and bone morphogenic protein 1). Mutations in the CUB region have been found in congenital TTP, but deletion of the region did not impair enzyme activity in conventional in vitro assays. We investigated the functions of the CUB domain in ADAMTS13 activity under flow conditions. We found that recombinant CUB-1 and CUB-1+2 polypeptides and synthetic peptides derived from CUB-1 partially blocked the cleavage of ULVWF by ADAMTS13 on the surface of endothelial cells under flow. The polypeptide bound immobilized and soluble forms of ULVWF, and blocked the adhesion of ADAMTS13-coated beads to immobilized ULVWF under flow. These results suggest that the CUB-1 domain may serve as the docking site for ADAMTS13 to bind ULVWF under flow, a critical step to initiate ULVWF proteolysis.
Using heparinized whole blood and flow conditions, it was shown that adenosine 5'-diphosphate (ADP) receptors P2Y(12) and P2Y(1) are both important in direct shear-induced platelet aggregation and ...platelet aggregation subsequent to initial adhesion onto von Willebrand factor (vWf)-collagen. In the viscometer, whole blood was subjected to shear rates of 750, 1500, and 3000 s(-1) for 30 seconds at room temperature. The extent of aggregation was determined by flow cytometry. The P2Y(12) antagonist AR-C69 931MX (ARMX) reduced shear-induced aggregation at these rates by 56%, 54%, and 16%, respectively, compared to control samples. Adenosine 3',5'-diphosphate (A3P5P; P2Y(1) antagonist) inhibited shear-induced aggregation by 40%, 30% and 29%, respectively, compared to control samples. Blockade of both ADP receptors at 3000 s(-1) with ARMX plus A3P5P further reduced the platelet aggregation by 41% compared to the addition of ARMX alone (57% compared to control samples). Using a parallel-plate flow chamber, whole blood was perfused over bovine collagen type 1 at a wall shear rate of 3000 s(-1) for 60 seconds. Platelet deposition was quantified with epifluorescence video microscopy and digital image processing. Blockade of P2Y(12) alone or blockade of P2Y(1) alone did not reduce thrombus formation on vWf-collagen. In contrast, blockade of both P2Y(12) and P2Y(1) reduced platelet deposition by 72%. These results indicate that combinations of antagonists of the ADP receptors P2Y(12) and P2Y(1) are effective inhibitors of direct shear-induced platelet aggregation and of platelet aggregation subsequent to initial adhesion under flow conditions. Inhibitors of these pathways are potentially useful as antiarterial thrombotic agents.
p53-mediated apoptosis may involve the induction of redox-controlling genes, resulting in the production of reactive oxygen species. Microarray expression analysis of doxorubicin exposed, related ...human lymphoblasts, p53 wild-type (WT) Tk6, and p53 mutant WTK1 identified the p53-dependent up-regulation of manganese superoxide dismutase (MnSOD) and glutathione peroxidase 1 (GPx). Consensus p53 binding sequences were identified in human MnSOD and GPx promoter regions. A 3-fold increase in the MnSOD promoter activity was observed after the induction of p53 in Li-Fraumeni syndrome (LFS) fibroblast, TR9-7, expressing p53 under the control of a tetracycline-regulated promoter. An increased protein expression of endogenous MnSOD and GPx also positively correlated with the level of p53 induction in TR9-7 cells. However, catalase (CAT) protein expression remained unaltered after p53 induction. We also examined the expression of MnSOD, GPx, and CAT in a panel of normal or LFS fibroblasts, containing either WT or mutant p53. We found increased MnSOD enzymatic activity, MnSOD mRNA expression, and MnSOD and GPx protein in LFS fibroblasts carrying a WT p53 allele when compared with homozygous mutant p53 isogenic cells. The CAT protein level was unchanged in these cells. We observed both the release of cytochrome C and Ca(2+) from the mitochondria into the cytoplasm and an increased frequency of apoptotic cells after p53 induction in the TR9-7 cells that coincided with an increased expression of MnSOD and GPx, and the level of reactive oxygen species. The increase in apoptosis was reduced by the antioxidant N-acetylcysteine. These results identify a novel mechanism of p53-dependent apoptosis in which p53-mediated up-regulation of MnSOD and GPx, but not CAT, produces an imbalance in antioxidant enzymes and oxidative stress.
Two likely mechanisms for the initiation of arterial platelet thrombus formation under conditions of elevated fluid shear stresses are: (1) excessive adhesion and aggregation of platelets from ...rapidly flowing blood onto the exposed subendothelium of injured, atherosclerotic arteries; or (2) direct, fluid shear stress-induced aggregation of platelets in constricted arteries with intact endothelial cells. Mechanism (1) was simulated using a parallel plate flow chamber, fibrillar collagen type l-coated slides, and mepacrine-labeled (fluorescent) platelets in whole blood anticoagulated with citrate, hirudin, unfractionated porcine heparin, or low molecular weight heparin flowing for 1 to 2 minutes at wall shear rates of 100 to 3,000 seconds−1 (4 to 120 dynes/cm2). The precise sequence of interactions among von Willebrand factor (vWF), glycoprotein (GP)lb, and GPIIb-IIIa during platelet adhesion and subsequent aggregation were resolved by direct real-time observation using a computerized epifluorescence video microscopy system. Adhesion at high shear rates was the result of the adsorption of large vWF multimers onto collagen and the binding of platelet GPIb to the insolubilized vWF. Aggregation occurred subsequently and required the binding of ligands, including vWF via its RGD binding domain, to GPIIb-IIIa. Mechanism (2) was modeled by producing shear stresses of 90 to 180 dynes/cm2 in a rotational cone and plate viscometer, which aggregates platelets from platelet-rich-plasma (PRP) anticoagulated with citrate, hirudin, or either type of heparin in reactions that require large vWF multimers, Ca2+, adenosine diphosphate, and both GPIb and GPIIb-IIIa. Both vWF-mediated shear-aggregation in PRP and platelet-collagen adhesion in flowing whole blood (anticoagulated with citrate and hirudin) are inhibited by two potentially useful anti-arterial thrombotic agents: polymeric aurin tricarboxylic acid (ATA; 28.5 to 114 µg/mL), which binds to vWF and inhibits its attachment of GPIb, and a recombinant vWF fragment (rvWF445-733; 30 to 200 µg/mL) that binds to platelet GPIb (in the absence of any modulator) and blocks attachment of vWF multimers. Unfractionated heparin, but not low molecular weight heparin, apparently binds to rvWF445-733 and counteracts the inhibitory effects of the vWF fragment in vitro on shear-aggregation and platelet-collagen adhesion.
In thrombotic thrombocytopenic purpura (TTP), a multimeric form of von
Willebrand factor (vWf) that is larger than ordinarily found in the plasma
causes systemic platelet aggregation under the ...high-shear conditions of the
microcirculation. A divalent cation-activated, vWf-cleaving
metalloprotease that metabolizes large vWf multimers to smaller forms in normal
plasma is severely reduced or absent in most patients with TTP. The
vWf-cleaving metalloprotease either is not produced or is defective in children
with chronic relapsing TTP. When the enzyme is provided by the infusion of
normal plasma, these patients remain free of TTP symptoms for about three
weeks. An IgG autoantibody to the vWf-cleaving metalloprotease is found
transiently in many adult patients with acute idiopathic, recurrent, and
ticlopidine/clopidogrel-associated TTP. These patients require plasma exchange,
i.e., concurrent replacement of the inhibited vWf-cleaving metalloprotease by
plasma infusion and plasmapheresis. The vWf-cleaving metalloprotease is present
in fresh-frozen plasma, in cryoprecipitate-depleted plasma (cryosupernatant),
and in plasma that has been treated with solvent and detergent. The
pathophysiology of platelet aggregation in bone marrow
transplantation/chemotherapy-associated thrombotic microangiopathy, and
in the hemolytic-uremic syndrome, is not established. In neither condition is
there a severe decrease in plasma vWf-cleaving metalloprotease
activity.
The metalloprotease ADAMTS‐13 cleaves von Willebrand factor (VWF) at the Y842/M843 peptide bond located in the A2 domain. Measurement of ADAMTS‐13 activity is a clinical utility for thrombotic ...diseases, but the current assays used for diagnostic and clinical research are non‐physiological and time consuming. We have expressed in bacteria a recombinant VWF‐A2 peptide (aa 718–905) that contains both a 6xHis tag at the N‐terminal end and a Tag‐100 epitope at the C‐terminal end. Diluted plasma was mixed with the VWF‐A2 peptide and digestion was allowed to proceed in a Ni2+‐coated microtiter well plate for 2 h. The immobilized Ni2+ captures the VWF‐A2 peptide by its 6xHis tag and cleavage of the A2 peptide is measured by the removal of the C‐terminus fragment of the A2 peptide that contains the Tag‐100. The cleavage activity for this assay was defined by the low detection of A2 peptide containing the Tag‐100 epitope by the antiTag‐100 monoclonal antibody. The assay was completed in <5 h. We then used the assay to analyze ADAMTS‐13 activity in plasma from 39 healthy donors and 16 samples from patients diagnosed as thrombotic thrombocytopenic purpura. The average of enzyme activity ± SEM for normal plasmas diluted 1 : 50 was 40 ± 4.2% while the value obtained for the patients was 2.4 ± 0.7%. These results were validated by a traditional long incubation assay (24 h). Our assay provides significant advantages over currently used assays because it is quicker, reproducible, cost effective and measures ADAMTS‐13 activity under physiological and non‐denaturing conditions. This assay is clinically useful and significant in measuring ADAMTS‐13 activity in plasma.
ADAMTS13 cleaves ultralarge and hyperreactive von Willebrand factor (ULVWF) freshly released from activated endothelial cells to smaller and less active forms. This process may be affected by the ...amount of ULVWF released and the processing capacity of ADAMTS13, contributing to the development of thrombotic diseases. We examined the effects of inflammatory cytokines on the release and cleavage of ULVWF to evaluate potential links between inflammation and thrombosis. Human umbilical vein endothelial cells were treated with interleukin 6 (IL-6), IL-8, or tumor necrosis factor α (TNF-α), and the formation of platelet-decorated ULVWF strings was quantitated. IL-8 and TNF-α significantly stimulated the release of ULVWF in a dose-dependent manner. IL-6 induced ULVWF release only when it was in complex with the soluble IL-6 receptor. IL-6, but not IL-8 nor TNF-α, inhibited the cleavage of ULVWF strings by ADAMTS13 under flowing, but not static, conditions. These results suggest that inflammatory cytokines may stimulate the ULVWF release (IL-8 and TNF-α) and inhibit the ULVWF cleavage (IL-6), resulting in the accumulation of hyperreactive ULVWF in plasma and on the surface of endothelial cells to induce platelet aggregation and adhesion on the vascular endothelium. The findings describe a potential linkage between inflammation and thrombosis that may be of therapeutic importance.
Platelets subjected to elevated levels of fluid shear stress in the absence of exogenous agonists will aggregate. Shear stress-induced aggregation requires von Willebrand factor (vWF) multimers, ...extracellular calcium (Ca2+), adenosine diphosphate (ADP), and platelet membrane glycoprotein (GP)Ib and GPIIb-IIIa. The sequence of interaction of vWF multimers with platelet surface receptors and the effect of these interactions on platelet activation have not been determined. To elucidate the mechanism of shear stress-induced platelet aggregation, suspensions of washed platelets were subjected to different levels of uniform shear stress (15 to 120 dyne/cm2) in an optically modified cone and plate viscometer. Cytoplasmic ionized calcium (Ca2+i) and aggregation of platelets were monitored simultaneously during the application of shear stress; Ca2+i was measured using indo-1 loaded platelets and aggregation was measured as changes in light transmission. Basal Ca2+i was approximately 60 to 100 nmol/L. An increase of Ca2+i (up to greater than 1,000 nmol/L) was accompanied by synchronous aggregation, and both responses were dependent on the shear force and the presence of vWF multimers. EGTA chelation of extracellular Ca2+ completely inhibited vWF-mediated Ca2+i and aggregation responses to shear stress. Aurin tricarboxylic acid, which blocks the GPIb recognition site on the vWF monomer, and 6D1, a monoclonal antibody to GPIb, also completely inhibited platelet responses to shear stress. The tetrapeptide RGDS and the monoclonal antibody 10E5, which inhibit vWF binding to GPIIb-IIIa, partially inhibited shear stress-induced Ca2+i and aggregation responses. The combination of creatine phosphate/creatine phosphokinase, which converts ADP to adenosine triphosphate and blocks the effect of ADP released from stimulated platelets, inhibited shear stress-induced platelet aggregation without affecting the increase of Ca2+i. Neither the Ca2+i nor aggregation response to shear stress was inhibited by blocking platelet cyclooxygenase metabolism with acetylsalicylic acid. These results indicate that GPIb and extracellular Ca2+ are absolutely required for vWF-mediated Ca2+i and aggregation responses to imposed shear stress, and that the interaction of vWF multimers with GPIIb-IIIa potentiates these responses. Shear stress-induced elevation of platelet Ca2+i, but not aggregation, is independent of the effects of release ADP, and both responses occur independently of platelet cyclooxygenase metabolism. These results suggest that shear stress induces the binding of vWF multimers to platelet GPIb and this vWF-GPIb interaction causes an increase of Ca2+i and platelet aggregation, both of which are potentiated by vWF binding to the platelet GPIIb-IIIa complex.