Antiplatelet treatment is of fundamental importance in combatting functions/dysfunction of platelets in the pathogenesis of cardiovascular and inflammatory diseases. Dysfunction of anucleate platelets is likely to be completely attributable to alterations in posttranslational modifications and protein expression. We therefore examined the proteome of platelets highly purified from fresh blood donations, using elaborate protocols to ensure negligible contamination by leukocytes, erythrocytes, and plasma. Using quantitative mass spectrometry, we created the first comprehensive and quantitative human platelet proteome, comprising almost 4000 unique proteins, estimated copy numbers for ∼ 3700 of those, and assessed intersubject (4 donors) as well as intrasubject (3 different blood samples from 1 donor) variations of the proteome. For the first time, our data allow for a systematic and weighted appraisal of protein networks and pathways in human platelets, and indicate the feasibility of differential and comprehensive proteome analyses from small blood donations. Because 85% of the platelet proteome shows no variation between healthy donors, this study represents the starting point for disease-oriented platelet proteomics. In the near future, comprehensive and quantitative comparisons between normal and well-defined dysfunctional platelets, or between platelets obtained from donors at various stages of chronic cardiovascular and inflammatory diseases will be feasible.

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.

Vasodilator-stimulated phosphoprotein (VASP), a substrate of cAMP- and cGMP-dependent protein kinases, is associated with focal adhesions, cell–cell contacts, microfilaments, and highly dynamic membrane regions. VASP, which is expressed in most cell types and in particularly high levels in human platelets, binds to profilin, zyxin, vinculin, F-actin, and theListeria monocytogenessurface protein ActA. VASP is a member of the enabled (Ena)/VASP protein family and is thought to be involved in actin filament formation and integrin α_IIbβ₃inhibition in human platelets. To gain further insight into thein vivofunction of this protein, VASP-deficient mice were generated by homologous recombination. VASP−/− mice demonstrated hyperplasia of megakaryocytes in bone marrow and spleen but exhibited no other macroscopic or microscopic abnormalities. Activation of platelets with thrombin induced a more than 2-fold higher surface expression of P-selectin and fibrinogen binding in VASP-deficient platelets in comparison to wild type. These data support the concept that VASP is a negative modulator of platelet and integrin α_IIbβ₃activation. Although the limited phenotypic differences between wild-type and VASP−/− mice suggested functional compensation of VASP by members of the Ena/VASP family, alterations in the expression levels of mammalian enabled (Mena) and Ena-VASP-like (Evl) protein were not detected. VASP-deficient mice may provide an interesting model system for diseases in which enhanced platelet activation plays a major role.

Protein kinase A (PKA) activation by cAMP phosphorylates multiple target proteins in numerous platelet inhibitory pathways that have a very important role in maintaining circulating platelets in a resting state. Here we show that in thrombin- and collagen-stimulated platelets, PKA is activated by cAMP-independent mechanisms involving dissociation of the catalytic subunit of PKA (PKAc) from an NFkappaB-IkappaBalpha-PKAc complex. We demonstrate mRNA and protein expression for most of the NFkappaB family members in platelets. From resting platelets, PKAc was co-immunoprecipitated with IkappaBalpha, and conversely, IkappaBalpha was also co-immunoprecipitated with PKAc. This interaction was significantly reduced in thrombin- and collagen-stimulated platelets. Stimulation of platelets with thrombin- or collagen-activated IKK, at least partly by PI3 kinase-dependent pathways, leading to phosphorylation of IkappaBalpha, disruption of an IkappaBalpha-PKAc complex, and release of free, active PKAc, which phosphorylated VASP and other PKA substrates. IKK inhibitor inhibited thrombin-stimulated IkBalpha phosphorylation, PKA-IkBalpha dissociation, and VASP phosphorylation, and potentiated integrin alphaIIbbeta3 activation and the early phase of platelet aggregation. We conclude that thrombin and collagen not only cause platelet activation but also appear to fine-tune this response by initiating downstream NFkappaB-dependent PKAc activation, as a novel feedback inhibitory signaling mechanism for preventing undesired platelet activation.