Leticia Nolasco

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.

N-acetylcysteine reduces the size and activity of von Willebrand factor in human plasma and mice

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease characterized by systemic microvascular thrombosis caused by adhesion of platelets to ultra-large vWF (ULVWF) multimers. These multimers accumulate because of a deficiency of the processing enzyme ADAMTS13. vWF protein forms long multimers from homodimers that first form through C-terminal disulfide bonds and then join through their N termini by further disulfide bonding. N-acetylcysteine (NAC) is an FDA-approved drug that has long been used to treat chronic obstructive lung disease and acetaminophen toxicity and is known to function in the former disorder by reducing mucin multimers. Here, we examined whether NAC could reduce vWF multimers, which polymerize in a manner similar to mucins. In vitro, NAC reduced soluble plasma-type vWF multimers in a concentration-dependent manner and rapidly degraded ULVWF multimer strings extruded from activated ECs. The effect was preceded by reduction of the intrachain disulfide bond encompassing the platelet-binding A1 domain. NAC also inhibited vWF-dependent platelet aggregation and collagen binding. Injection of NAC into ADAMTS13-deficient mice led to the rapid resolution of thrombi produced by ionophore treatment of the mesenteric venules and reduced plasma vWF multimers. These results suggest that NAC may be a rapid and effective treatment for patients with TTP.

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

Summary.  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/cm2 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.

Platelet‐derived VWF‐cleaving metalloprotease ADAMTS‐13

Summary.  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.

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/μl. 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 α‐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 α‐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. Am. J. Hematol. 57:293–302, 1998.

Endothelial cell ADAMTS-13 and VWF: production, release, and VWF string cleavage

Human umbilical vein endothelial cell (HUVEC)-released ADAMTS-13 (a disintegrin and metalloprotease with thrombospondin repeats) and HUVEC-secreted von Willebrand factor (VWF) strings were investigated under static conditions that allow the accumulation and analysis of ADAMTS-13. The latter was released constitutively from HUVECs and cleaved the secreted and cell-anchored VWF strings progressively during 15 minutes in Ca(2+)/Zn(2+)-containing buffer. HUVEC ADAMTS13 mRNA expression was approximately 1:100 of VWF monomeric subunit expression. In contrast to multimeric VWF stored within Weibel-Palade bodies and secreted rapidly in response to cell stimulation, ADAMTS-13 was released directly from the Golgi to the cell exterior without an organelle storage site. The constitutive release of ADAMTS-13 continued at the same slow rate regardless of the presence or absence of histamine stimulation of HUVECs. Consequently, the percentage of VWF strings cleaved by ADAMTS-13 at VWF Y(1605)-M(1606) decreased as the rate of VWF string secretion was increased by cell stimulation. Blockade of HUVEC ADAMTS-13 activity by antibodies to different ADAMTS-13 domains made it possible to detect the attachment of ADAMTS-13 all along the lengths of HUVEC-secreted VWF strings. Constitutive ADAMTS-13 released from endothelial cells may contribute to the maintenance of cell surfaces free of hyperadhesive VWF multimeric strings.

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

Summary.  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.

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

Summary.  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.

COVALENT REGULATION OF ULVWF STRING FORMATION AND ELONGATION ON ENDOTHELIAL CELLS UNDER FLOW CONDITIONS

Summary.  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‐PEO2‐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.

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); and35S-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 (300–360×103/μl) 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.