Platelets play a crucial role in the physiology of primary hemostasis and pathophysiologic processes such as arterial thrombosis. Accumulating evidence suggests a role of reactive oxygen species (ROSs) in platelet activation. Here we show that platelets activated with different agonists produced intracellular ROSs, which were reduced by reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidase inhibitors and superoxide scavengers. In addition, we demonstrate that ROSs produced in platelets significantly affected αIIbβ3 integrin activation but not alpha and dense granule secretion and platelet shape change. Thrombin-induced integrin αIIbβ3 activation was significantly decreased after pretreatment of platelets with NAD(P)H oxidase inhibitors (diphenylene iodonium [DPI] [45% ± 9%] and apocynin [43% ± 11%]) and superoxide scavengers (tiron [60% ± 9%] and Mn(III)tetrakis (1-methyl-4-pyridyl)porphyrin [MnTMPyP] [70% ± 6%]). These inhibitors also reduced platelet aggregation and thrombus formation on collagen under high shear and achieved their effects independent of the nitric oxide/cyclic guanosine monophosphate (NO/cGMP) pathway.

Filamin A (FlnA) cross-links actin filaments and connects the Von Willebrand factor receptor GPIb-IX-V to the underlying cytoskeleton in platelets. Because FlnA deficiency is embryonic lethal, mice lacking FlnA in platelets were generated by breeding FlnAloxP/loxP females with GATA1-Cre males. FlnAloxP/y GATA1-Cre males have a macrothrombocytopenia and increased tail bleeding times. FlnA-null platelets have decreased expression and altered surface distribution of GPIbα because they lack the normal cytoskeletal linkage of GPIbα to underlying actin filaments. This results in ∼70% less platelet coverage on collagen-coated surfaces at shear rates of 1,500/s, compared with wild-type platelets. Unexpectedly, however, immunoreceptor tyrosine-based activation motif (ITAM)- and ITAM-like–mediated signals are severely compromised in FlnA-null platelets. FlnA-null platelets fail to spread and have decreased α-granule secretion, integrin αIIbβ3 activation, and protein tyrosine phosphorylation, particularly that of the protein tyrosine kinase Syk and phospholipase C–γ2, in response to stimulation through the collagen receptor GPVI and the C-type lectin-like receptor 2. This signaling defect was traced to the loss of a novel FlnA–Syk interaction, as Syk binds to FlnA at immunoglobulin-like repeat 5. Our findings reveal that the interaction between FlnA and Syk regulates ITAM- and ITAM-like–containing receptor signaling and platelet function.

Filamin A (FlnA) is a large cytoplasmic protein that crosslinks actin filaments and anchors membrane receptors and signaling intermediates. FlnAloxP PF4-Cre mice that lack FlnA in the megakaryocyte (MK) lineage have a severe macrothrombocytopenia because of accelerated platelet clearance. Macrophage ablation by injection of clodronate-encapsulated liposomes increases blood platelet counts in FlnAloxP PF4-Cre mice and reveals the desintegration of FlnA-null platelets into microvesicles, a process that occurs spontaneously during storage. FlnAloxP PF4-Cre bone marrows and spleens have a 2.5- to 5-fold increase in MK numbers, indicating increased thrombopoiesis in vivo. Analysis of platelet production in vitro reveals that FlnA-null MKs prematurely convert their cytoplasm into large CD61+ platelet-sized particles, reminiscent of the large platelets observed in vivo. FlnA stabilizes the platelet von Willebrand factor receptor, as surface expression of von Willebrand factor receptor components is normal on FlnA-null MKs but decreased on FlnA-null platelets. Further, FlnA-null platelets contain multiple GPIbα degradation products and have increased expression of the ADAM17 and MMP9 metalloproteinases. Together, the findings indicate that FlnA-null MKs prematurely release large and fragile platelets that are removed rapidly from the circulation by macrophages.

Platelets play a key role in hemostasis through their ability to rapidly adhere to activated or injured endothelium, subendothelial matrix proteins, and other activated platelets. A strong equilibrium between activating and inhibiting processes is essential for normal platelet and vascular function, impairment of this equilibrium being associated with either thrombophilic or bleeding disorders. Both cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) have been established as crucial and synergistic intracellular messengers that mediate the effects of platelet inhibitors such as nitric oxide (NO) and prostacyclin (PG-I2). However, it was recently suggested that a rapid cGMP/cGMP-dependent protein kinase (cGK)–mediated extracellular signal-related kinase (ERK) phosphorylation promotes platelet activation. This hypothesis was examined here by evaluating established and proposed cGK activators/inhibitors with respect to their capacity to promote either platelet activation or inhibition. In particular, the regulatory role of cGK for ERK phosphorylation and thrombin-, thromboxane-, and VWF-induced platelet activation was investigated. The data obtained do not support the concept that cGK-mediated ERK phosphorylation promotes platelet activation but confirm the inhibitory role of cGK in platelet function. One explanation for these discrepancies is the novel finding that extracellular cGMP analogs potently and rapidly inhibit thrombin-, thromboxane-, and VWF-induced human platelet signaling and activation by a cGK-independent mechanism.