Plasma fibronectin promotes thrombus growth and stability in injured arterioles

Mice lacking both of the best-known platelet ligands, von Willebrand factor and fibrinogen, can still form occlusive thrombi in injured arterioles. The platelets of these animals accumulate excessive amounts of fibronectin (FN). These observations led us to examine the contribution of plasma FN (pFN) to thrombus formation. Inactivation of the FN gene in FN conditional knockout mice reduced pFN levels to <2% and platelet FN to ≈20% of the levels in similarly treated control mice. The mice were then observed in a model of arterial injury to evaluate their capacity to form thrombi. The deficiency of pFN did not affect the initial platelet adhesion, but a delay of several minutes in thrombus formation was observed in the arterioles of pFN-deficient mice as compared with control mice. The thrombi that formed in the absence of pFN were stably anchored to the vessel wall but continuously shed platelets or small platelet clumps, thus slowing their growth significantly; the platelet/platelet cohesion was apparently diminished. Consequently the occlusion of pFN-deficient vessels was delayed, with the majority of vessels remaining patent at the end of the 40-min observation period. We conclude that, in addition to von Willebrand factor and fibrinogen, FN plays a significant role in thrombus initiation, growth, and stability at arterial shear rates and that deficiency in each of the three platelet ligands has its own specific impact on platelet plug formation

The Aryl hydrocarbon receptor is activated by modified low-density lipoprotein

Endogenous activation of the aryl hydrocarbon receptor (AHR) is required for normal vascular development. This biology led us to investigate the interplay between the AHR and vascular physiology by using an in vitro model of fluid shear stress. Using this system, we show that fluid flow induces a robust AHR-mediated increase in CYP1 expression. Furthermore, we demonstrate that incubation with sheared bovine or human sera is sufficient for AHR activation, indicating that direct cellular exposure to shear stress is not required for this response. Fractionation of sera by size and density revealed the AHR-activating factor to be low-density lipoprotein (LDL). Purified LDL (0.1 mg/ml) from sheared sera induces a 6-fold increase in AHR-mediated signaling as compared with LDL purified from static sera. Similar results were obtained by exposing a purified fraction of LDL to fluid flow, suggesting that shear stress is capable of directly modifying LDL structure and/or function. In addition, we show that LDL can be converted to an AHR-activating species by conventional methods of lipoprotein modification, such as NaOCl oxidation. Finally, we demonstrate that an increased level of AHR-activating LDL is present in the sera of AHR null mice as compared with heterozygous littermates, suggesting a role for the Ahr locus in the physiological response to modified LDL in vivo. Overall, these data demonstrate a previously undescribed relationship between LDL modification and AHR biology and provide a potential explanation for the vascular abnormalities observed in AHR null mice

Functional self-association of von Willebrand factor during platelet adhesion under flow

We have used recombinant wild-type human von Willebrand factor (VWF) and deletion mutants lacking the A1 and A3 domains, as well as specific function-blocking monoclonal antibodies, to demonstrate a functionally relevant self-association at the interface of soluble and surface-bound VWF. Platelets perfused at the wall shear rate of 1,500 s(−1) over immobilized VWF lacking A1 domain function failed to become tethered to the surface when they were in a plasma-free suspension with erythrocytes, but adhered promptly if soluble VWF with functional A1 domain was added to the cells. The same results were observed when VWF was immobilized onto collagen through its A3 domain and soluble VWF with deleted A3 domain was added to the cells. Thus, VWF bound to glass or collagen sustains a process of homotypic self-association with soluble VWF multimers that, as a result, can mediate platelet adhesion. The latter finding demonstrates that direct immobilization on a substrate is not a strict requirement for VWF binding to platelet glycoprotein Ibα. The dynamic and reversible interaction of surface-bound and soluble VWF appears to be specifically homotypic, because immobilized BSA, human fibrinogen, and fibronectin cannot substitute for VWF in the process. Our findings highlight a newly recognized role of circulating VWF in the initiation of platelet adhesion. The self-assembly of VWF multimers on an injured vascular surface may provide a relevant contribution to the arrest of flowing platelets opposing hemodynamic forces, thus facilitating subsequent thrombus growth