Aubrey Bernardo

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.

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.

Von Willebrand factor present in fibrillar collagen enhances platelet adhesion to collagen and collagen-induced platelet aggregation.

Summary.  We examined the basis of the differences observed between different collagen preparations in their ability to aggregate platelets and support their adhesion under flow. As in previous studies, we found fibrillar collagen to be 10‐fold more potent than acid‐soluble collagen in inducing platelet aggregation and found that acid‐soluble collagen did not support the adhesion of washed platelets under flow. Further, platelets in whole blood adhered to surfaces coated with either fibrillar or acid‐soluble collagen, but thrombi formed faster and grew larger on fibrillar collagen. As a possible basis for this difference, we found that fibrillar collagen, but not acid‐soluble collagen, contains a substantial quantity of von Willebrand factor (VWF), as demonstrated by enzyme‐linked immunosorbent assay and by the ability of fibrillar collagen to support the adhesion of VWF antibody‐coated beads and to agglutinate GPIb–IX–V complex‐expressing Chinese hamster ovary cells. Supporting a role for VWF in collagen‐induced platelet aggregation, aggregation induced by acid‐soluble collagen was greatly enhanced by added VWF. Further, platelet aggregation by fibrillar collagen was partially blocked by a GPIbα antibody that inhibits the GPIb–VWF interaction. Taken together, these results suggest that much of the difference in prothrombotic potency of different collagens is directly related to their differences in VWF content. This probably accounts for the different conclusions made regarding the relative importance of different direct and indirect collagen receptors in collagen‐dependent platelet functions and further emphasizes the close synergistic roles of the GPIb–IX–V complex and the collagen receptors GPVI and α2β1 in supporting platelet adhesion.

Variations among normal individuals in the cleavage of endothelial-derived ultra-large von Willebrand factor under flow.

Summary.  von Willebrand factor (VWF) freshly released from endothelial cells is normally cleaved by the ADAMTS‐13 metalloprotease to prevent the direct release of these ultra‐large (UL) and hyper‐reactive multimers into plasma. The balance of ULVWF proteolysis may be regulated by the amount of ULVWF released and the processing capacity of ADAMTS‐13. The former associates with the size of ULVWF storage pool, sensitivity of vascular endothelial cells to stimulation, and the type of agonists, whereas the latter associates with the activity of ADAMTS‐13. These parameters may vary significantly among individuals. We have determined the variations of ADAMTS‐13 activity in 68 normal individuals by a flow‐based assay and a static assay using ULVWF strings and recombinant VWF A2 domain as substrates, respectively. We found that the levels of ADAMTS‐13 activity required to cleave the platelet‐decorated ULVWF strings under flow is significantly higher than that of static assays. Normal plasma diluted to 25% significantly reduced its ability to cleave ULVWF strings under flow, whereas 2% plasma retained 48% enzyme activity in static assay. ADAMTS‐13 activity varied from 33 to 100% among individuals and the variations were greater at shorter incubations of plasma with the substrate. Furthermore, the production of ULVWF from endothelial cells also varied among individuals. These results suggest that the commonly used static assays may underestimate the ADAMTS‐13 activity required to cleave newly released ULVWF. They also demonstrated that the proteolysis of ULVWF may vary significantly among individuals, potentially contributing to the individuals vulnerability to thrombosis so that measurement of ADAMTS‐13 may serve as a marker for TTP and other thrombotic diseases.

Shear stress augments the enhanced adhesive phenotype of cells expressing the Pro33 isoform of integrin β3

Adhesion of platelets to the exposed extracellular matrix proteins at sites of vascular injury is partly regulated by the local fluid shear stress. Because the Leu33Pro (PlA) polymorphism of integrin β3 confers only a modest increase in adhesion under static conditions, we used CHO and 293 cells expressing the Leu33 or Pro33 isoform of β3 in flow chamber experiments to test whether shear forces would alter the PlA adhesive phenotype. We found that shear force augmented the Pro33‐mediated enhanced adhesion to fibrinogen. This Pro33‐dependent enhancement was aspirin‐sensitive and was also observed on immobilized von Willebrand factor and cryoprecipitate, but not fibronectin. Thus, shear stress enhances the adhesive phenotype of the Pro33 cells to multiple physiologic substrates.

A G-quartet oligonucleotide blocks glycoprotein Ib-mediated platelet adhesion and aggregation under flow conditions

Platelets arrest bleeding by adhering to and aggregating on the subendothelium exposed at the site of vessel injury. This process is initiated by the interaction between the subendothelium von Willebrand factor (VWF) and the glycoprotein (GP) Ib-IX-V complex on platelets. However, the same interaction also results in thrombosis at the site of a ruptured atherosclerotic plaque. Reagents regulating the GP Ib-VWF interaction will therefore have direct impact on haemostasis and thrombosis. We have characterised an oligonucleotide G-quartet (T30923) that specifically blocks VWF binding to GP Ibalpha, the VWF-binding subunit of the GP Ib-IX-V complex. We evaluated the potential interactions of T30923 with GP Ibalpha and VWF A1 domain by computer simulated molecular dockings, which identified four T30923 docking sites in the beta-sheets of the N-terminal region of GP Ibalpha (E14-D18, S39, D63-S64, and D83-S85). Experimentally, T30923 bound GP Ibalpha and dose-dependently blocked platelet aggregation induced by ristocetin and thrombin, but not by botrocetin, collagen, TRAP, and ADP. It also blocked shear-induced platelet aggregation and thrombus formation on immobilised VWF under arterial shear stress. These results demonstrate that T30923 may have therapeutic potentials to regulate the GP Ibalpha-VWF interaction.