Platelets are anucleate blood cells, long known to be critically involved in hemostasis and thrombosis. In addition to their role in blood clots, increasing evidence reveals significant roles for platelets in inflammation and immunity. However, the notion that platelets represent immune cells is not broadly recognized in the field of Physiology. This manuscript reviews the role of platelets in inflammation and immune responses, and highlights their interactions with other immune cells, including examples of major functional consequences of these interactions.

Genetic factors are believed to influence the development of arterial thromboses. Because integrin α IIb β 3 plays a crucial role in thrombus formation, we analyzed receptor adhesive properties using Chinese hamster ovary and human kidney embryonal 293 cells overexpressing the Pl A1 or Pl A2 polymorphic forms of α IIb β 3 . Soluble fibrinogen binding was no different between Pl A1 and Pl A2 cells, either in a resting state or when α IIb β 3 was activated with anti-LIBS6. Pl A1 and Pl A2 cells bound equivalently to immobilized fibronectin. In contrast, significantly more Pl A2 cells bound to immobilized fibrinogen in an α IIb β 3 -dependent manner than did Pl A1 cells. Disruption of the actin cytoskeleton by cytochalasin D abolished the increased binding of Pl A2 cells. Compared with Pl A1 cells, Pl A2 cells exhibited a greater extent of polymerized actin and cell spreading, enhanced tyrosine phosphorylation of pp125 FAK , and greater fibrin clot retraction. These adhesion differences appear to depend on a signaling mechanism sensitive to receptor occupancy. Thus, the Pl A2 polymorphism altered integrinmediated functions of adhesion, spreading, actin cytoskeleton rearrangement, and clot retraction. Generation of cell lines. The cDNA for β 3 (19) corresponding to the Pl A1 polymorphism was mutated to code for the Pl A2 polymorphism using the Altered Sites II in vitro Mutagenesis Kit (Promega Corp., Madison, Wisconsin, USA) as described previously (20) and sequenced to confirm authenticity. The cDNAs for α IIb (21) and for β 3 (either Pl A1 or Pl A2 ) were engineered into the LK444 vector (22) and used to transfect CHO cells. In addition, the cDNA for α IIb was engineered into the pZeoSV2 vector (Invitrogen Corp., Carlsbad, California, USA) and the cDNA for β 3 (either Pl A1 or Pl A2 ) engineered into the pcDNA3.1 vector (Invitrogen Corp.) and used to transfect 293 cells. Both CHO and 293 cells were transfected using lipofectin (Life Technologies Inc., Gaithersburg, Maryland, USA) (23) with the respective plasmids for both α IIb and β 3 subunits. Control CHO cells (designated LK444) were transfected 794The Flow cytometry. CHO and 293 cells were grown to 70-80% confluency and detached using 0.05% trypsin (Life Technologies). After neutralization with complete media, the cells were suspended in PBS (pH = 7.4; 0.137 M NaCl, 4.3 mM Na 2 HPO 4 , 1.4 mM KH 2 PO 4, 2.7 mM KCl) with 2% BSA and incubated with either 3 µg/mL P2, 2 µg/mL anti-β 3 mAb (clone SZ21; Immunotech), or 3 µg/mL control mouse IgG (Pierce Chemical Co., Rockford, Illinois, USA) for 1 hour on ice. The cells were washed, incubated with a secondary goat antimouse FITC-labeled antibody, and analyzed on a FACScalibur flow cytometer (Becton Dickinson, San Jose, California, USA), with fluorescence data acquired in the logarithmic mode and light scattering data acquired in the linear mode (24). The mean channel number, which corresponded to cell-fluorescence intensity, was used as the measure of surface-expressed α IIb β 3 for the whole population.Western blot ...

Background Platelet hyperactivity induced by inflammation is a known risk factor for atherosclerosis and thrombosis, but its underlying mechanisms remain poorly understood. Methods and Results The signal transducers and activators of transcription 3 (STAT3) was activated in collagen-stimulated platelets. Activated STAT3 served as a protein scaffold to facilitate the catalytic interaction between the kinase Syk and the substrate PLCγ2 to enhance collagen-induced calcium mobilization and platelet activation. The same interaction of STAT3 with Syk and PLCγ2 was also detected in HEK293 cells transfected with cDNAs for Syk and PLCγ2, and stimulated with interleukin-6 (IL-6). Pharmacological inhibition of STAT3 blocked ~50% of collagen- and a collagen-related peptide-, but not TRAP- or ADP-induced aggregation and ~80% of thrombus formation of human platelets on a collagen matrix. This in vitro phenotype was reproduced in mice infused with STAT3 inhibitors and mice with platelet specific STAT3 deficiency. By forming a complex with its soluble receptor, the proinflammatory cytokine IL-6 enhanced the collagen-induced STAT3 activation in human platelets. Conclusions These data demonstrate a non-transcriptional activity of STAT3 that facilitates a crosstalk between proinflammatory cytokine and hemostasis/thrombosis signals in platelets. This crosstalk may be responsible for platelet hyperactivity found in conditions of inflammation.

This study used recombinant A1A2A3 tri-domain proteins to demonstrate that A domain association in von Willebrand factor (VWF) regulates the binding to platelet glycoprotein Ib␣ (GPIb␣). We performed comparative studies between wild type (WT) A1 domain and the R1450E variant that dissociates the tri-domain complex by destabilizing the A1 domain. Using urea denaturation and differential scanning calorimetry, we demonstrated the destabilization of the A1 domain structure concomitantly results in a reduced interaction among the three A domains. This dissociation results in spontaneous binding of R1450E to GPIb␣ without ristocetin with an apparent K D of 85 ؎ 34 nM, comparable with that of WT (36 ؎ 12 nM) with ristocetin. The mutant blocked 100% ristocetin-induced platelet agglutination, whereas WT failed to inhibit. The mutant enhanced shear-induced platelet aggregation at 500 and 5000 s ؊1 shear rates, reaching 42 and 66%, respectively. Shear-induced platelet aggregation did not exceed 18% in the presence of WT. A1A2A3 variants were added before perfusion over a fibrin(ogen)-coated surface. At 1500 s ؊1 , platelets from blood containing WT adhered <10% of the surface area, whereas the mutant induced platelets to rapidly bind, covering 100% of the fibrin(ogen) surface area. Comparable results were obtained with multimeric VWF when ristocetin (0.5 mg/ml) was added to blood before perfusion. EDTA or antibodies against GPIb␣ and ␣IIb␤3 blocked the effect of the mutant and ristocetin on platelet activation/adhesion. Therefore, the termination of A domain association within VWF in solution results in binding to GPIba and platelet activation under high shear stress.Platelet adhesion at sites of vascular injury contributes to the arrest of bleeding as well as to the pathologic occlusion of diseased vessels under elevated shear stress. Under this high shear stress, the platelet-von Willebrand factor (VWF) 3 interaction is essential for platelet adhesion. The interaction between VWF and the exposed subendothelium permits the VWF to interact with circulating platelets via the receptor glycoprotein (GP)Ib/ IX/V complex (1). Mature VWF consists of a 2050-residue subunit that contains domains that are arranged in the order DЈ-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK (cystine knot) (2-4). The VWF contains a triplicate repeat sequence or A domains in the central portion of the subunit. Binding sites for platelet GPIb␣, heparin, sulfatides, and collagen (5-9) are within the A1 domain, whereas its homologous A3 domain only binds to collagen, and the A2 domain contains the cleavage site for the metalloprotease ADAMTS-13 (10 -12). The functions of these A domains relevant to the biology of VWF have been characterized by individual recombinant expression of each of the A domains (9, 10, 13).The interaction between VWF and GPIb␣ occurs when the binding site for GPIb␣ in the A1 domain of VWF has been exposed by the influence of high fluid forces or when the multimeric protein has been immobilized (14). This activation can also be induced by nat...