Glutathione regulates integrin alpha(IIb)beta(3)-mediated cell adhesion under flow conditions.

The platelet integrin alpha(IIb)beta(3) mediates the final step of platelet aggregation that requires pre-activation through an inside-out signal initiated by agonists. Experiments conducted under static conditions using platelet-rich plasma show that platelet activation and adhesion activity of alpha(IIb)beta(3) are regulated by glutathione (GSH-GSSG) redox potential. However, it remains unclear as to whether GSH-GSSG exerts its regulatory role in platelets by direct targeting of alpha(IIb)beta(3) or intracellular signals that activate the integrin. A role of fluid shear stress is also not known. We examined the effects of GSH-GSSG on the adhesion of CHO cells expressing two HPA variants of human alpha(IIb)beta(3) to the immobilized fibrinogen and von Willebrand factor (VWF) under flow conditions. GSH-GSSG dose-dependently reduced the number of adherent cells to fibrinogen and VWF under 2.5 dyn/cm(2) of shear stress, a physical force calculated to be 110 dyne on platelets. GSH treatment also abolished the hyper-adhesion activity of cells expressing the Pro33 variant of alpha(IIb)beta(3). The inhibition was also observed with washed platelets. The data differ from the early observation that GSH enhanced platelet aggregation induced by sub-threshold concentrations of platelet agonists. The results suggest that GSH may have distinct effects on agonist-induced alpha(IIb)beta(3) activation and on the alpha(IIb)beta(3)-fibrinogen or alpha(IIb)beta(3)-VWF bonds when exposed to fluid shear stress. They further suggest that the HPA phenotype may be redox-regulated.

Acquired ADAMTS-13 deficiency in pediatric patients with severe sepsis.

We studied the state of ultra-large von Willebrand factor (ULVWF) proteolysis in 21 pediatric patients with severe sepsis and found that the overall group of patients had moderately reduced ADAMTS-13 activity, but 31% had severe enzymatic deficiency. The severe deficiency correlated with greater adhesion activity of von Willebrand factor, severity of thrombocytopenia and plasma levels of interleukin-6. It also correlated clinically with severity of illness and organ dysfunction. These results suggest that ULVWF proteolysis is insufficient in septic patients and severely deficient in a subgroup of patients. The deficiency may contribute to the development of thrombocytopenia and ischemic organ failure associated with sepsis.

Effects of inflammatory cytokines on the release and cleavage of the endothelial cell-derived ultralarge von Willebrand factor multimers under flow.

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 alpha (TNF-alpha), and the formation of platelet-decorated ULVWF strings was quantitated. IL-8 and TNF-alpha 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-alpha, 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-alpha) 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.

ADAMTS-13 metalloprotease interacts with the endothelial cell-derived ultra-large von Willebrand factor.

Thrombotic thrombocytopenic purpura is caused by congenital or acquired deficiency of ADAMTS-13, a metalloprotease that cleaves the endothelium-derived ultra-large multimers of von Willebrand factor (ULVWF). The proteolysis converts hyper-reactive and thrombogenic ULVWF into smaller and less adhesive plasma forms. Activity of ADAMTS-13 is usually measured in a static system under non-physiological conditions that require protein denaturation and prolonged incubation. We have demonstrated previously that ULVWF multimers, upon release from endothelial cells, form platelet-decorated string-like structures that are rapidly cleaved by ADAMTS-13. Here we report the direct interaction between ADAMTS-13 and VWF under both static and flowing conditions. ADAMTS-13-coated beads adhered to both immobilized VWF and ULVWF strings presented by stimulated endothelial cells. These beads adhered to VWF under both venous (2.5 dynes/cm2) and arterial (30 dynes/cm2) shear stresses. We then demonstrated that ADAMTS-13 beads adhered to immobilized recombinant VWF-A1 and -A3 domains, but soluble metalloprotease bound preferentially to the A3 domain, suggesting that the VWF A3 domain may be the primary docking site for the metalloprotease. We suggest that tensile stresses imposed by fluid shear stretch endothelial bound ULVWF multimers to expose binding sites within the A domains for circulating ADAMTS-13. The bound enzyme then cleaves within the A2 domain that lies in close proximity and releases smaller VWF multimers into the plasma. Once released, these cleaved VWF fragments become inaccessible for the metalloprotease to prevent further cleavage.

Effects of low temperature on shear-induced platelet aggregation and activation.

BACKGROUND: Hemorrhage is a major complication of trauma and often becomes more severe in hypothermic patients. Although it has been known that platelets are activated in the cold, studies have been focused on platelet behavior at 4 degrees C, which is far below temperatures encountered in hypothermic trauma patients. In contrast, how platelets function at temperatures that are commonly found in hypothermic trauma patients (32-37 degrees C) remains largely unknown, especially when they are exposed to significant changes in fluid shear stress that could occur in trauma patients due to hemorrhage, vascular dilation/constriction, and fluid resuscitation. METHODS: Using a cone-plate viscometer, we have examined platelet activation and aggregation in response to a wide range of fluid shear stresses at 24, 32, 35, and 37 degrees C. RESULTS: We found that shear-induced platelet aggregation was significantly increased at 24, 32, and 35 degrees C as compared with 37 degrees C and the enhancement was observed in whole blood and platelet-rich plasma. In contrast to observation made at 4 degrees C, the increased shear-induced platelet aggregation at these temperatures was associated with minimal platelet activation as determined by the P-selectin expression on platelet surface. Blood viscosity was also increased at low temperature and the changes in viscosity correlated with levels of plasma total protein and fibrinogen. CONCLUSION: We found that platelets are hyper-reactive to fluid shear stress at temperatures of 24, 32, and 35 degrees C as compared with at 37 degrees C. The hyperreactivity results in heightened aggregation through a platelet-activation independent mechanism. The enhanced platelet aggregation parallels with increased whole blood viscosity at these temperatures, suggesting that enhanced mechanical cross-linking may be responsible for the enhanced platelet aggregation.

P-selectin anchors newly released ultralarge von Willebrand factor multimers to the endothelial cell surface.

von Willebrand factor (VWF) released from endothelium is ultralarge (UL) and hyperreactive. If released directly into plasma, it can spontaneously aggregate platelets, resulting in systemic thrombosis. This disastrous consequence is prevented by the ADAMTS13 (ADisintegrin and Metalloprotease with ThromboSpondin motif) cleavage of ULVWF into smaller, less active forms. We previously showed that ULVWF, on release, forms extremely long stringlike structures. ADAMTS13 cleaves these strings under flow significantly faster than it does under static conditions. As ULVWF tethering to endothelium is important for its rapid proteolysis, we investigated 2 molecules for their potential to anchor the ULVWF strings: P-selectin and integrin alpha v beta 3. We demonstrated that P-selectin anchors ULVWF to endothelium by several means. First, Chinese hamster ovary (CHO) cells expressing P-selectin specifically adhered to immobilized ULVWF and ULVWF-coated beads to immobilized P-selectin. Second, an anti-VWF antibody coimmunoprecipitates P-selectin from the histamine-activated endothelial cells. Third, P-selectin antibody or soluble P-selectin, but not a alpha v beta 3 antibody, RGDS peptide, or heparin, blocked the formation of ULVWF strings. Fourth, P-selectin expression was in clusters predominantly along the ULVWF strings. Finally, the strength of the minimal ULVWF-P-selectin bond was measured to be 7.2 pN. We, therefore, conclude that P-selectin may anchor ULVWF strings to endothelial cells and facilitate their cleavage by ADAMTS13.