Von Willebrand factor activates endothelial nitric oxide synthase in blood platelets by a glycoprotein Ib-dependent mechanism.
BACKGROUND
The molecular regulation of endothelial nitric oxide synthase (eNOS) in blood platelets and the signalling events induced by platelet-derived NO are poorly defined. In particular, the ability of von Willebrand factor (VWF) to stimulate cyclic guanosine monophosphate (cGMP) formation in platelets has produced conflicting data.


OBJECTIVE
To determine the mechanisms leading to eNOS activation and clarify the downstream signaling pathways activated by platelet-derived NO in response to VWF.


METHODS
We used three independent markers of NO signaling, [3H] l-citrulline production, cGMP accrual and immunoblotting of vasodilator-stimulated phosphoprotein (VASP) to examine the NO signaling cascade in response to VWF.


RESULTS
VWF increased NO synthesis and bioavailability, as evidenced by increased [3H] l-citrulline production and cGMP accrual, respectively. VWF-induced eNOS activation was GPIb-IX-dependent and independent of integrin alpha(IIb)beta3. cGMP formation in response to VWF required Ca2+ mobilization, Src family kinases, phosphatidylinositol 3-kinase and phospholipase C, but not protein kinase C. This suggests that a cross-talk between the signaling mechanisms regulates platelet activation and NO synthesis. VWF-induced cGMP accrual was completely blocked by apyrase and indomethacin, demonstrating an essential role for platelet-derived ADP and thromboxane A2 (TxA2). Elevated cGMP levels led to increased VASP phosphorylation at serine239 that was both protein kinase G (PKG)- and protein kinase A (PKA)-dependent.


CONCLUSIONS
We demonstrate that VWF activates eNOS through a specific Ca2+-dependent GPIb receptor-signaling cascade that relies on the generation of platelet-derived ADP and TxA2. Furthermore, we provide the first evidence to suggest that platelet derived-NO/cGMP activates PKA in addition to PKG.
Riba
R
R
Medical Biosciences, University of Bradford, Bradford, West Yorkshire, UK.
Oberprieler
N G
NG
Roberts
W
W
Naseem
K M
KM
D000244:Adenosine Diphosphate / Q000378:metabolism; D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008840:Microfilament Proteins / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051966:Phospholipase C gamma / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D015539:Platelet Activation; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism*; D015398:Signal Transduction; D013928:Thromboxane A2 / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism; D014841:von Willebrand Factor / Q000378:metabolism*; D000098662:Vasodilator-Stimulated Phosphoprotein
D015815:Cell Adhesion Molecules; D008840:Microfilament Proteins; D010750:Phosphoproteins; D019038:Platelet Glycoprotein GPIb-IX Complex; D000098662:Vasodilator-Stimulated Phosphoprotein; D014841:von Willebrand Factor; D013928:Thromboxane A2; D000244:Adenosine Diphosphate; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D019061:src-Family Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D051966:Phospholipase C gamma; D006152:Cyclic GMP; D002118:Calcium