Hiuwan Choi

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.

Shear-induced Disulfide Bond Formation Regulates Adhesion Activity of von Willebrand Factor

von Willebrand factor (VWF) is the largest multimeric adhesion ligand circulating in blood. Its adhesion activity is related to multimer size, with the ultra-large forms freshly released from the activated endothelial cells being most active, capable of spontaneously binding to platelets. In comparison, smaller plasma forms circulating in blood bind platelets only under high fluid shear stress or induced by modulators. The structure-function relationships that distinguish the two types of VWF multimers are not known. In this study, we demonstrate that some of the plasma VWF multimers contain surface-exposed free thiols. Physiological and pathological levels of shear stresses (50 and 100 dynes/cm2) promote the formation of disulfide bonds utilizing these free thiols. The shear-induced thiol-disulfide exchange increases VWF binding to platelets. The thiol-disulfide exchange involves some or all of nine cysteine residues (Cys889, Cys898, Cys2448, Cys2451, Cys2490, Cys2491, Cys2453, Cys2528, and Cys2533) in the D3 and C domains as determined by mass spectrometry of the tryptic VWF peptides. These results suggest that the thiol-disulfide state may serve as an important structural determinant of VWF adhesion activity and can be modified by fluid shear stress.

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.

Disulfide Bond Reduction of Von Willebrand Factor by ADAMTS-13

See also Lenting PJ, Rastegarlari G. ADAMTS‐13: double trouble for von Willebrand factor. This issue, pp 2775–7.

Antihemostatic Activity of Human Granzyme B Mediated by Cleavage of von Willebrand Factor

The cytotoxic lymphocyte protease granzyme B (GrB) is elevated in the plasma of individuals with diseases that elicit a cytotoxic lymphocyte-mediated immune response. Given the recently recognized ability of GrB to cleave extracellular matrix proteins, we examined the effect of GrB on the pro-hemostatic molecule von Willebrand factor (VWF). GrB delays ristocetin-induced platelet aggregation and inhibits platelet adhesion and spreading on immobilized VWF under static conditions. It efficiently cleaves VWF at two sites within the A1–3 domains that are essential for the VWF-platelet interaction. Like the VWF regulatory proteinase ADAMTS-13, GrB-mediated cleavage is dependent upon VWF conformation. In vitro, GrB cannot cleave the VWF conformer found in solution, but cleavage is induced when VWF is artificially unfolded or presented as a matrix. GrB cleaves VWF with comparable efficiency to ADAMTS-13 and rapidly processes ultra-large VWF multimers released from activated endothelial cells under physiological shear. GrB also cleaves the matrix form of fibrinogen at several sites. These studies suggest extracellular GrB may help control localized coagulation during inflammation.

Magnesium maintains endothelial integrity, up-regulates proteolysis of ultra-large von Willebrand factor, and reduces platelet aggregation under flow conditions

Mg (++) regulates endothelial functions and has anti-inflammatory effects. Its effects on thrombosis have been demonstrated, but the mechanism remains poorly understood. We investigated the roles of MgSO(4) in regulating the release and cleavage of the prothrombotic ultra-large (UL) von Willebrand factor (VWF) and VWF-mediated platelet adhesion and aggregation. Washed platelets were perfused over cultured endothelial cells from human umbilical cord veins under a shear stress of 2.5 dyn/cm(2). Release and cleavage of ULVWF by ADAMTS-13 was measured in the absence or presence of physiological or therapeutic levels of MgSO(4). Whole blood or plasma-free reconstituted blood was perfused over immobilized collagen to measure the effect of MgSO(4) on platelet adhesion and aggregation. Also studied were the effects of MgSO(4) on ristocetin-induced platelet aggregation andVWF-collagen interaction. Maintenance of endothelial integrity required physiological levels of MgSO(4), but exogenous MgSO(4) showed no additional benefits. Exogenous MgSO(4) significantly enhanced the cleavage of the newly released ULVWF strings by ADAMTS-13 and markedly reduced platelet aggregation on immobilized collagen under flow conditions. This effect is likely to be mediated through VWF as Mg(++) partially inhibited ristocetin-induced platelet aggregation and VWF binding to collagen. MgSO(4) is critical for maintaining endothelial integrity and regulates ULVWF proteolysis and aggregation under flow conditions. These results provide a new insight into additional mechanisms involved with magnesium therapy.