Protein phosphatase 2B inhibition promotes the secretion of von Willebrand factor from endothelial cells.

BACKGROUND: Secretion of Weibel-Palade body (WPB) contents is regulated, in part, by the phosphorylation of proteins that constitute the endothelial exocytotic machinery. In comparison to protein kinases, a role for protein phosphatases in regulating endothelial exocytosis is undefined. OBJECTIVE AND METHOD: In this study, we investigated the role of protein phosphatase 2B (PP2B) in the process of endothelial exocytosis using pharmacological and gene knockdown approaches. RESULTS: We show that inhibition of protein phosphatase 2B (PP2B) activity by cyclosporine A (CsA), tacrolimus or a cell-permeable PP2B autoinhibitory peptide promotes the secretion of ultralarge von Willebrand factor (ULVWF) from human umbilical vein endothelial cells (HUVECs) in the absence of any other endothelial cell-stimulating agent. PP2B inhibitor-induced secretion and anchorage of ULVWF strings from HUVECs mediate platelet tethering. In support of a role for PP2B in von Willebrand factor (VWF) secretion, the catalytic subunit of PP2B interacts with the vesicle trafficking protein, Munc18c. Serine phosphorylation of Munc18c, which promotes granule exocytosis in other secretory cells, is increased in CsA-treated HUVECs, suggesting that this process may be involved in CsA-mediated WPB exocytosis. Furthermore, the plasma VWF antigen level is also enhanced in CsA-treated mice, and small interfering RNA-mediated knockdown of the alpha and beta isoforms of the PP2B-A subunit in HUVECs enhanced VWF secretion. CONCLUSIONS: These observations suggest that CsA promotes VWF release, in part by inhibition of PP2B activity, and are compatible with the clinically observed association of CsA treatment and increased plasma VWF levels in humans.

Covalent regulation of ULVWF string formation and elongation on endothelial cells under flow conditions.

BACKGROUND AND OBJECTIVES: The adhesion ligand von Willebrand factor (VWF) is a multimeric glycoprotein that mediates platelet adhesion to exposed subendothelium. On endothelial cells, freshly released ultra-large (UL) VWF multimers form long string-like structures to which platelets adhere. METHODS: The formation and elongation of ULVWF strings were studied in the presence of the thiol-blocking N-ethylmaleimide (NEM). The presence of thiols in ULVWF and plasma VWF multimers was determined by maleimide-PEO(2)-Biotin labeling and thiol-chromatography. Finally, covalent re-multimerization of ULVWF was examined in a cell- and enzyme-free system. RESULTS: We found that purified plasma VWF multimers adhere to and elongate ULVWF strings under flow conditions. The formation and propagation of ULVWF strings were dose-dependently reduced by blocking thiols on VWF with NEM, indicating that ULVWF strings are formed by the covalent association of perfused VWF to ULVWF anchored to endothelial cells. The association is made possible by the presence of free thiols in VWF multimers and by the ability of (UL) VWF to covalently re-multimerize. CONCLUSION: The data provide a mechanism by which the thrombogenic ULVWF strings are formed and elongated on endothelial cells. This mechanism suggests that the thiol-disulfide state of ULVWF regulates the adhesion properties of strings on endothelial cells.

Novel ADAMTS-13 mutations in an adult with delayed onset thrombotic thrombocytopenic purpura.

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP) is associated with congenital and acquired deficiency of ADAMTS-13, a metalloprotease that cleaves von Willebrand factor (VWF) and reduces its adhesive activity. Mutations throughout the ADAMTS13 gene have been identified in congenital TTP patients, most of whom have initial episodes during infancy or in early childhood. PATIENTS AND METHODS: We report the case of an adult male who was diagnosed with idiopathic thrombocytopenic purpura at age 34, and with TTP 14 years later. The patient was compound heterozygous for an 18 bp in-frame deletion (C365del) in the disintegrin domain and a point mutation of R1060W in the seventh thrombospondin domain of the ADAMTS-13 gene. CONCLUSIONS: In vitro studies found that C365del and R1060W severely impair ADAMTS-13 synthesis in transfected Hela cells, whereas the deletion mutant also failed to cleave VWF under static and flow conditions.

Human endothelial cells synthesize and release ADAMTS-13.

Hepatic stellate cells have been considered to be a primary source for human plasma ADAMTS-13, the von Willebrand factor (VWF)-cleaving metalloprotease. In this study, ADAMTS-13 antigen was detected by immunofluorescence in both venous (HUVECs) and arterial endothelial cells (HUAECs) using both polyclonal antibodies made against peptides found in various domains of human ADAMTS-13, as well as by a monoclonal antibody against the ADAMTS-13 metalloprotease domain. ADAMTS-13 antigen had an intra-cellular distribution in endothelial cells distinct from the Weibel-Palade body location of VWF, and was released from the cells during 48 h in culture. The mRNA for ADAMTS13 was detected in HUVECs and HUAECs using reverse transcription-polymerase chain reaction (RT-PCR), indicating that the enzyme is synthesized in these cells. The ADAMTS-13 protein was immunoprecipitated from HUVECs and had an approximate M(r) of 170 kDa, similar to the molecular mass of recombinant ADAMTS-13. The ADAMTS-13 in HUVEC and HUAEC lysates had enzymatic activity using both static and flow assays. We conclude that ADAMTS-13 is synthesized in human endothelial cells, and released constitutively. The vast number of endothelial cells in the body may be an important source of ADAMTS-13.

Platelet-derived VWF-cleaving metalloprotease ADAMTS-13.

The adhesion ligand von Willebrand factor (VWF) is synthesized and stored in vascular endothelial cells and megakaryocytes/platelets. As in endothelial cells, platelet VWF also contains ultra-large (UL) multimers that are hyperactive in aggregating platelets. ULVWF in platelet lysates of thrombin-stimulated platelets was only detected in the presence of EDTA, suggesting that ULVWF is cleaved by a divalent cation-dependent protease. A recent study shows that platelets contain the VWF-cleaving metalloprotease ADAMTS-13, but its activity remains unknown. In this study, we show that platelet lysates cleave endothelial cell-derived ULVWF under static and flow conditions. This activity is inhibited by EDTA and by an ADAMTS-13 antibody from the plasma of a patient with acquired TTP. ADAMTS-13 was detected in platelet lysates and on the platelet surface by four antibodies that bind to different domains of the metalloprotease. Expression of ADAMTS-13 on the platelet surface increases significantly upon platelet activation by the thrombin receptor-activating peptide, but not by ADP. These results demonstrate that platelets contain functionally active ADAMTS-13. This intrinsic activity may be physiologically important to prevent the sudden release of hyperactive ULVWF from platelets and serves as the second pool of ADAMTS-13 to encounter the increase in ULVWF release from endothelial cells.

Platelets adhered to endothelial cell-bound ultra-large von Willebrand factor strings support leukocyte tethering and rolling under high shear stress.

Leukocyte rolling on vascular endothelium is mediated by an interaction between P-selectin expressed on endothelial cells and P-selectin glycoprotein ligand-1 on leukocytes. This interaction reduces the velocity of leukocyte movements to allow subsequent firm adhesion and transmigration. However, the interaction has so far been observed only under low venous shear stress and cannot explain the accumulation of monocytes in atherosclerotic plaques found in arteries, where shear stress is much higher. We have previously shown that newly released ultra-large von Willebrand factor (ULVWF) forms extremely long string-like structures to which platelets tether. Here, we investigated whether platelets adhered to ULVWF strings are activated and form aggregates. We also determined whether activated platelets on ULVWF strings can support leukocyte tethering and rolling under high shear stresses. We found that platelets adhered to ULVWF expressed P-selectin and bound PAC-1, suggesting their rapid activation. We also found that leukocytes tethered to and rolled on these platelet-decorated ULVWF strings, but not directly on endothelial cells, under high shear stresses of 20 and 40 dyn/cm(2) in a P-selectin dependent manner. These results suggest that the endothelial cell-bound ULVWF provide an ideal matrix to aggregate platelets and recruit leukocytes to endothelial cells under high shear stress. The observed phenomenon delineates a mechanism for leukocytes to be tethered to arterial endothelial cells under high shear, providing a potential link between inflammation and thrombosis.

ADAMTS-13 activity in plasma is rapidly measured by a new ELISA method that uses recombinant VWF-A2 domain as substrate.

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.

Platyspondylic lethal skeletal dysplasia, San Diego type, is caused by FGFR3 mutations.

The platyspondylic lethal skeletal dysplasias (PLSDs) are a heterogeneous group of short-limb dwarfing conditions. The most common form of PLSD is thanatophoric dysplasia (TD), which has been divided into two types (TD1 and TD2). Three other types of PLSD, or TD variants (San Diego, Torrance, and Luton), have been distinguished from TD. The most notable difference between TD and the variants is the presence of large rough endoplasmic reticulum (rER) inclusion bodies within chondrocytes of the variants. We examined 22 cases of TD variants for the presence of missense mutations in the fibroblast growth factor receptor 3 (FGFR3) gene. All 17 cases of the San Diego type (PLSD-SD) were heterozygous for the same FGFR3 mutations found in TD1. No mutations were identified in the Torrance and Luton types. Large inclusion bodies were found in all 14 cases of PLSD-SD. Similar inclusion bodies were present in two of 72 TD1 cases, but not in 39 controls. The material retained within the rER stained only with antibody to the FGFR3 protein. The radiographic and morphologic differences between TD and PLSD-SD may be a consequence of other genetic factors, perhaps in the processing of mutant FGFR3 molecules within the rER. The presence of rER inclusion bodies cannot reliably discriminate between closely related skeletal dysplasias.

Lethal osteosclerotic osteochondrodysplasia with platyspondyly, metaphyseal widening, and intracellular inclusions in sibs.

We report on a previously undescribed form of lethal osteosclerotic skeletal dysplasia in sibs from nonconsanguineous parents. Radiographic findings included increased density in the base of the skull, clavicles, vertebrae, ribs, and the metaphyseal regions of the long bones. There was midface hypoplasia, a large anterior fontanel, micrognathia, and hypoplastic, wafer-thin vertebrae. The clavicles, ribs, metacarpals, metatarsals, and phalanges were especially thickened and widened. The long bones were shortened with flared metaphyses. Chondroosseous morphology of resting cartilage and growth plate was relatively normal, but there was hypercellular cortical and trabecular bone, and marrow fibrosis. Ultrastructurally, the resting chondrocytes, osteoblasts, and nonhematopoietic marrow cells had dilated rough endoplasmic reticulum (inclusion bodies). The radiographic and morphologic characteristics in this case are unique and differ from those seen in other previously reported lethal osteosclerotic skeletal dysplasias.

Increased von Willebrand factor binding to platelets in single episode and recurrent types of thrombotic thrombocytopenic purpura.

Extensive microvascular platelet aggregation is characteristic of thrombotic thrombocytopenic purpura (TTP). Previous studies have indicated that abnormalities of von Willebrand factor (vWf) are often present in TTP patient plasma. There has not been previously any direct evidence linking these abnormalities to the process of intravascular platelet aggregation in TTP. We used flow cytometry to analyze the binding of vWf to single platelets, and the presence of platelet aggregates, in the blood of 4 children with chronic relapsing (CR) TTP and 5 adults with single episode or recurrent TTP. vWf on the single platelets of CRTTP patients at all time points studied was significantly increased compared to controls, and was increased further as platelet counts decreased to levels below 40,000/microl. The single episode and recurrent adult TTP patients had platelet aggregates in the blood, as well as increased vWf on single platelets, before therapy commenced and thereafter until recovery was in process. In the one unresponsive single episode TTP patient, vWf on single platelets remained elevated, and platelet aggregates persisted, until her death. The platelet alpha-granular protein, P-selectin, was not increased on the single platelets of most TTP blood samples, suggesting that it is vWf from plasma (rather than from alpha-granules) that attaches to platelet surfaces in association with platelet aggregation. These results suggest that vWf-platelet interactions are involved in the platelet clumping process that characterizes TTP.

Direct demonstration of radiolabeled von Willebrand factor binding to platelet glycoprotein Ib and IIb-IIIa in the presence of shear stress.

In this study it is demonstrated for the first time that shear stress induces the binding of exogenous von Willebrand factor (vWF) multimers to platelets. The vWF preparations used were: 125I-vWF purified from human cryoprecipitate (and including all vWF multimers present in normal plasma); and 35S-cysteine-vWF secreted by human umbilical vein endothelial cells (HUVECs) (and containing unusually large vWF forms, as well as all plasma-type vWF multimers). Direct shear-induced binding to washed platelets (300-360 x 10(3)/microliters) of radiolabeled vWF was maximum at 60-120 dynes/cm2 evaluated at 30 sec and was in extent about one-quarter of the binding stimulated by ristocetin after 3 min of incubation. The shear-induced binding of only a small percentage of added radiolabeled vWF was sufficient to initiate aggregation. Radiolabeled vWF attached to both glycoprotein (GP) Ib and GPIIb-IIIa receptors in the shear field, with complete inhibition of binding occurring with simultaneous blockade of both receptors. Binding was potentiated by ADP released from sheared platelets.

Solvent/detergent-treated plasma suppresses shear-induced platelet aggregation and prevents episodes of thrombotic thrombocytopenic purpura.

Two children with congenital chronic relapsing thrombotic thrombocytopenic purpura (TTP) have episodes every 3 weeks. These relapses can be prevented by the infusion of normal fresh-frozen plasma (FFP) without concurrent plasmapheresis. We conducted a study to determine whether the exposure of normal plasma to agents that inactivate human immunodeficiency virus and other viruses destroys the component necessary for the effective treatment of this type of TTP that requires only plasma infusion to prevent or reverse relapses. Clinical responsiveness and von Willebrand factor (vWF)-mediated fluid shear stress-induced platelet aggregation were evaluated before and after the infusion of 10 mL/kg FFP or solvent [tri(n-butyl)phosphate]/detergent (Triton X-100)-treated plasma (S/D plasma). Platelet aggregation at shear stresses of 90 to 180 dyne/cm2 (similar to those in the partially occluded microcirculation) imposed for 30 seconds was excessive using the citrated platelet-rich plasma of both patients, and was associated with the presence of unusually large vWF forms in patient platelet-poor plasma. Infusion with either FFP or S/D plasma at 3-week intervals caused the platelet count to increase to (or above) normal within 1 week (on 12 of 12 occasions); the disappearance or diminution of unusually large vWF forms within 1 hour (on 6 of 10 occasions studied); and the reversal within 1 to 4 hours of excessive shear-induced platelet aggregation (on 8 of 9 occasions studied). We conclude that a component in normal plasma resistant to S/D treatment is responsible for preventing thrombocytopenia and TTP episodes, and for controlling excessive shear-induced aggregation in these patients. Our results suggest that excessive in vivo platelet aggregation in chronic relapsing TTP and excessive in vitro vWF-mediated shear-induced aggregation may be similar phenomena.

Granulocyte proteases do not process endothelial cell-derived unusually large von Willebrand factor multimers to plasma vWF in vivo.

The unusually large von Willebrand factor (ULvWF) multimers present within endothelial cells and platelets are larger than the vWF multimers normally found in adult human plasma. Furthermore, ULvWF multimers are cleared rapidly from the circulation if they are released by intense endothelial cell stimulation. The mechanisms by which the ULvWF multimers are processed to large plasma vWF multimers are not known. It has been demonstrated that granulocyte proteases are capable of decreasing vWF multimer size in vitro, and that some patients with myeloproliferative syndromes have a relative absence of large plasma vWF multimers in sodium citrate-anticoagulated plasma samples. In order to assess the influence of granulocyte proteases on vWF multimer size, we evaluated the vWF multimeric patterns in 94 plasma samples from 60 patients with neutrophil counts that were either considerably elevated or extremely reduced. In 83 of 94 plasma samples, the vWF multimeric patterns were normal. No patients with very low neutrophil counts had ULvWF multimers present. These observations suggest that granulocyte proteases are not likely to be involved in vivo in the processing of ULvWF multimers from endothelial cells to the smaller vWF forms in circulation.

Cryosupernatant regulates accumulation of unusually large vWF multimers from endothelial cells.

In addition to the von Willebrand factor (vWF) multimers found in normal plasma, cultured human umbilical vein endothelial cells (HUVECs) synthesize and release unusually large vWF multimers (ULvWFM). ULvWFM are more effective than the largest plasma vWF forms in attaching to platelets and promoting platelet aggregation in the presence of elevated fluid shear forces (as in narrowed atherosclerotic vessels), and they may be involved in arterial thrombosis. ULvWFM are produced within HUVECs exposed for 48-60 h either to steady venous-like or pulsatile arterial-like wall shear stresses and are produced and released from HUVECs grown in serum-free as well as in serum (bovine or human)-containing media. An activity of 140,000-200,000 Da in the cryosupernatant fraction of both normal and severe von Willebrand's disease plasma, which is not obviously a protease, prevents specifically the accumulation of ULvWFM in the fluid above HUVEC monolayers but does not impair the release by HUVECs of ULvWFM in the retrograde direction into subendothelial collagen. The ULvWF regulatory activity in cryosupernatant may inhibit inappropriate platelet aggregation and thrombosis by preventing the accumulation of endothelial cell-derived ULvWFM in circulating blood.

Aurin tricarboxylic acid: a novel inhibitor of the association of von Willebrand factor and platelets.

Shear stress activated platelets undergo aggregation in the presence of large or unusually large von Willebrand factor (vWF) multimers without the addition of ristocetin or any other exogenous chemical. This phenomenon may be analogous to the platelet aggregation that leads to thrombosis in the narrowed arteries and arterioles of patients with atherosclerosis or vasospasm. A triphenyl-methyl compound, aurin tricarboxylic acid (ATA), inhibits shear-induced, vWF-mediated platelet aggregation in platelet-rich plasma (PRP) in concentrations above 200 mumol/L and in buffer suspensions of washed platelets at a concentration of 0.1 mumol/L. In a concentration-dependent manner, ATA also inhibits ristocetin-induced, vWF-mediated platelet clumping in both fresh and formaldehyde-fixed platelet suspensions. This inhibition can be overcome by increasing the concentration of vWF, following the kinetics of first order competitive inhibition. ATA prevents the attachment to platelets of the largest vWF multimeric forms found in normal plasma and of the unusually large vWF multimers derived from endothelial cells. The rate of aggregation and degree of inhibition by ATA is not accounted for by the binding of ristocetin or calcium. Arachidonic acid- and adenosine diphosphate (ADP)-induced aggregation are not inhibited by ATA. Platelets incubated with ATA can be easily separated from the compound. However, ATA binds to large vWF multimeric forms and inhibits their ristocetin-induced interaction with platelet glycoprotein Ib. Because ATA also inhibits shear-induced, vWF-mediated platelet aggregation in vitro in the absence of ristocetin, it may be a useful prototype compound to impede the development of arterial thrombosis in vivo.

Alkaline and acid phosphatase demonstration in human bone and cartilage: effects of fixation interval and methacrylate embedments.

Human bone and cartilage specimens were evaluated for acid and alkaline phosphatase localization following varying fixation periods for fresh or frozen tissue. Formalin fixations of up to 183 hr were followed by embedment in methyl methacrylate; frozen tissue was examined either without fixation or following fixation for up to 1 hr and subsequent glycol or methyl methacrylate embedding. The humeral epiphysis of a young patient with osteogenic sarcoma showed optimum acid and alkaline phosphatase localization following fixation for periods up to 15 hr and embedding in methyl methacrylate. Frozen costochondral junction from a newborn with osteogenesis imperfecta type II showed optimum acid and alkaline phosphatase localization following 30 min fixation in formalin and embedding in methyl methacrylate or after 5 min fixation and embedding in glycol methacrylate.

Shear-induced platelet aggregation can be mediated by vWF released from platelets, as well as by exogenous large or unusually large vWF multimers, requires adenosine diphosphate, and is resistant to aspirin.

Fluid shear stress in arteries and arterioles partially obstructed by atherosclerosis or spasm may exceed the normal time-average level of 20 dyne/cm2. In vitro, at fluid shear stresses of 30 to 60 dyne/cm2 applied for 30 seconds, platelet aggregation occurs. At these shear stresses, either large or unusually large von Willebrand factor (vWF) multimers in the suspending fluid exogenous to the platelets mediates aggregation. Adenosine diphosphate (ADP) is also required and, in these experiments, was released from the platelets subjected to shear stress. At 120 dyne/cm2, the release of endogenous platelet vWF multimers can substitute for exogenous large or unusually large vWF forms in mediating aggregation. Endogenous released platelet vWF forms, as well as exogenous large or unusually large vWF multimers, must bind to both glycoproteins Ib and the IIb/IIIa complex to produce aggregation. Shear-induced aggregation is the result of shear stress alteration of platelet surfaces, rather than of shear effects on vWF multimers. It is mediated by either large plasma-type vWF multimers, endogenous released platelet vWF forms, or unusually large vWF multimers derived from endothelial cells, requires ADP, and is not inhibited significantly by aspirin. This type of aggregation may be important in platelet thrombus formation within narrowed arterial vessels, and may explain the limited therapeutic utility of aspirin in arterial thrombosis.

Involvement of large plasma von Willebrand factor (vWF) multimers and unusually large vWF forms derived from endothelial cells in shear stress-induced platelet aggregation.

A fluid shear stress of 180 dyn/cm2 was applied for 0.5 and 5 min to platelets in citrated plasma or blood in a cone and plate viscometer with minimal platelet-surface interactions. Platelets aggregated in the shear field if large von Willebrand Factor (vWF) multimers were present. Aggregation did not require ristocetin, other exogenous agents, or desialation of vWF. Unusually large vWF multimers produced by human endothelial cells were functionally more effective than the largest plasma vWF forms in supporting shear-induced aggregation. Shear-induced aggregation was inhibited by monoclonal antibodies to platelet glycoprotein Ib or the IIb/IIIa complex, but was little affected by the absence of fibrinogen. vWF-dependent platelet aggregation under elevated shear stress in partially occluded vessels of the arterial microcirculation may contribute to thrombosis, especially if unusually large vWF multimers are released locally from stimulated or disrupted endothelial cells.