Comparison of head impact location during games and practices in Division III men's lacrosse players.

BACKGROUND: Head impacts have been studied extensively in football, but little similar research has been conducted in men's lacrosse. It is important to understand the location and magnitude of head impacts during men's lacrosse to recognize the risk of head injury. METHODS: Descriptive epidemiology study set on collegiate lacrosse fields. Eleven men's lacrosse players (age=20.9±1.13years, mass=83.91±9.04kg, height=179.88±5.99cm) volunteered to participate. We applied X2 sensors behind the right ear of participants for games and practices. Sensors recorded data on linear and rotational accelerations and the location of head impacts. We calculated incidence rates per 1000 exposures with 95% confidence intervals for impact locations and compared the effect of impact location on linear and rotational accelerations with Kruskal-Wallis tests. FINDINGS: We verified 167 head impacts (games=112; practices=55). During games, the incidence rate was 651.16 (95% confidence interval=530.57-771.76). The high and low incidence rates for head impact locations during games were: side=410.7 (95% confidence interval=292.02-529.41) and top=26.79 (95% confidence interval=3.53-57.10). For games and practices combined, the impact locations did not significantly affect linear (χ23=6.69, P=0.08) or rotational acceleration (χ23=6.34, P=0.10). INTERPRETATION: We suggest further research into the location of head impacts during games and practices. We also suggest player and coach education on head impacts as well as behavior modification in men's lacrosse athletes to reduce the incidence of impacts to the side of the head in an effort to reduce potential injury.

Identification of an antithrombotic allosteric modulator that acts through helix 8 of PAR1.

G protein-coupled receptors (GPCRs) can assume multiple conformations and possess multiple binding sites. Whereas endogenous agonists acting at the orthosteric binding site stabilize the active receptor conformation, small molecules that act at nonorthosteric sites can stabilize alternative conformations. The large majority of these allosteric modulators associate with extracellular loops of GPCRs. The role of intracellular domains in mediating allosteric modulation is largely unknown. In screening a small-molecule library for inhibitors of platelet activation, we identified a family of compounds that modified PAR1-mediated granule secretion. The most potent inhibitory compound, termed JF5, also demonstrated noncompetitive inhibition of the α(2A)-adrenergic receptor. Aggregation studies using a battery of platelet GPCR agonists demonstrated that sensitivity to JF5 was limited to GPCRs that possessed a constrained eighth helix, as defined by a C-terminal palmitoylation site and interactions with TM7 and the i1 loop. Inhibition by JF5 was overcome in a PAR1 mutant in which the eighth helix was deleted, confirming a role for helix 8 in JF5 activity. Evaluation of downstream signaling showed that JF5 was selective with regard to G protein coupling, blocking signaling mediated by G(αq) but not G(α12). The compound inhibited thrombus formation in vivo following vascular injury with an IC(50) of ∼1 mg/kg. These results indicate a role for helix 8 in conferring sensitivity to small molecules, and show that this sensitivity can be exploited to control platelet activation during thrombus formation.

Hyperglycemia-induced cerebral hematoma expansion is mediated by plasma kallikrein.

Hyperglycemia is associated with greater hematoma expansion and poor clinical outcomes after intracerebral hemorrhage. We show that cerebral hematoma expansion triggered by intracerebral infusion of autologous blood is greater in diabetic rats and mice compared to nondiabetic controls and that this augmented expansion is ameliorated by plasma kallikrein (PK) inhibition or deficiency. Intracerebral injection of purified PK augmented hematoma expansion in both diabetic and acutely hyperglycemic rats, whereas injection of bradykinin, plasmin or tissue plasminogen activator did not elicit such a response. This response, which occurs rapidly, was prevented by co-injection of the glycoprotein VI agonist convulxin and was mimicked by glycoprotein VI inhibition or deficiency, implicating an effect of PK on inhibiting platelet aggregation. We show that PK inhibits collagen-induced platelet aggregation by binding collagen, a response enhanced by elevated glucose concentrations. The effect of hyperglycemia on hematoma expansion and PK-mediated inhibition of platelet aggregation could be mimicked by infusing mannitol. These findings suggest that hyperglycemia augments cerebral hematoma expansion by PK-mediated osmotic-sensitive inhibition of hemostasis.

The platelet actin cytoskeleton associates with SNAREs and participates in alpha-granule secretion.

Following platelet activation, platelets undergo a dramatic shape change mediated by the actin cytoskeleton and accompanied by secretion of granule contents. While the actin cytoskeleton is thought to influence platelet granule secretion, the mechanism for this putative regulation is not known. We found that disruption of the actin cytoskeleton by latrunculin A inhibited alpha-granule secretion induced by several different platelet agonists without significantly affecting activation-induced platelet aggregation. In a cell-free secretory system, platelet cytosol was required for alpha-granule secretion. Inhibition of actin polymerization prevented alpha-granule secretion in this system, and purified platelet actin could substitute for platelet cytosol to support alpha-granule secretion. To determine whether SNAREs physically associate with the actin cytoskeleton, we isolated the Triton X-100 insoluble actin cytoskeleton from platelets. VAMP-8 and syntaxin-2 associated only with actin cytoskeletons of activated platelets. Syntaxin-4 and SNAP-23 associated with cytoskeletons isolated from either resting or activated platelets. When syntaxin-4 and SNAP-23 were tested for actin binding in a purified protein system, only syntaxin-4 associated directly with polymerized platelet actin. These data show that the platelet cytoskeleton interacts with select SNAREs and that actin polymerization facilitates alpha-granule release.

Immune versus thrombotic stimulation of platelets differentially regulates signalling pathways, intracellular protein-protein interactions, and alpha-granule release.

In addition to haemostasis, platelets mediate inflammation and clearance of bacteria from the bloodstream. As with platelet-platelet interactions, platelet-bacteria interactions involve cytoskeletal rearrangements and release of granular content. Stimulation of the immune Toll-like receptor 2 (TLR2) on the platelet surface, activates phosphoinositide-3-kinase (PI3K) and causes platelet activation and platelet-dependent thrombosis. It remains unknown if platelet activation by immune versus thrombotic pathways leads to the differential regulation of signal transduction, protein-protein interactions, and alpha-granule release, and the physiological relevance of these potential differences. We investigated these processes after immune versus thrombotic platelet stimulation. We examined selected signalling pathways and found that phosphorylation kinetics of Akt, ERK1/2 and p38 differed dramatically between agonists. Next, we investigated platelet protein-protein interactions by mass spectrometry (MS)-based proteomics specifically targeting cytosolic factor XIIIa (FXIIIa) because of its function as a cytoskeleton-crosslinking protein whose binding partners have limited characterisation. Four FXIIIa-binding proteins were identified, two of which are novel interactions: FXIIIa-focal adhesion kinase (FAK) and FXIIIa-gelsolin. The binding of FAK to FXIIIa was found to be altered differentially by immune versus thrombotic stimulation. Lastly, we studied the effect of thrombin versus Pam(3)CSK(4) stimulation on alpha-granule release and observed differential release patterns for selected granule proteins and decreased fibrin clot formation compared with thrombin. The inhibition of PI3K caused a decrease in protein release after Pam(3)CSK(4)- but not after thrombin-stimulation. In summary, stimulation of platelets by either thrombotic or immune receptors leads to markedly different signalling responses and granular protein release consistent with differential contribution to coagulation and thrombosis.

Platelet alpha-granules: basic biology and clinical correlates.

alpha-Granules are essential to normal platelet activity. These unusual secretory granules derive their cargo from both regulated secretory and endocytotic pathways in megakaryocytes. Rare, inheritable defects of alpha-granule formation in mice and man have enabled identification of proteins that mediate cargo trafficking and alpha-granule formation. In platelets, alpha-granules fuse with the plasma membrane upon activation, releasing their cargo and increasing platelet surface area. The mechanisms that control alpha-granule membrane fusion have begun to be elucidated at the molecular level. SNAREs and SNARE accessory proteins that control alpha-granule secretion have been identified. Proteomic studies demonstrate that hundreds of bioactive proteins are released from alpha-granules. This breadth of proteins implies a versatile functionality. While initially known primarily for their participation in thrombosis and hemostasis, the role of alpha-granules in inflammation, atherosclerosis, antimicrobial host defense, wound healing, angiogenesis, and malignancy has become increasingly appreciated as the function of platelets in the pathophysiology of these processes has been defined. This review will consider the formation, release, and physiologic roles of alpha-granules with special emphasis on work performed over the last decade.

Endobrevin/VAMP-8-dependent dense granule release mediates thrombus formation in vivo.

Individuals whose platelets lack dense or alpha-granules suffer various degrees of abnormal bleeding, implying that granule cargo contributes to hemostasis. Despite these clinical observations, little is known regarding the effects of impaired platelet granule secretion on thrombus formation in vivo. In platelets, SNARE proteins mediate the membrane fusion events required for granule cargo release. Endobrevin/VAMP-8 is the primary vesicle-SNARE (v-SNARE) responsible for efficient release of dense and alpha-granule contents; thus, VAMP-8(-/-) mice are a useful model to evaluate the importance of platelet granule secretion in thrombus formation. Thrombus formation, after laser-induced vascular injury, in these mice is delayed and decreased, but not absent. In contrast, thrombus formation is almost completely abolished in the mouse model of Hermansky-Pudlak syndrome, ruby-eye, which lacks dense granules. Evaluation of aggregation of VAMP-8(-/-) and ruby-eye platelets indicates that defective ADP release is the primary abnormality leading to impaired aggregation. These results demonstrate the importance of dense granule release even in the earliest phases of thrombus formation and validate the distal platelet secretory machinery as a potential target for antiplatelet therapies.

Balancing role of nitric oxide in complement-mediated activation of platelets from mCd59a and mCd59b double-knockout mice.

CD59 is a membrane protein inhibitor of the membrane attack complex (MAC) of complement. mCd59 knockout mice reportedly exhibit hemolytic anemia and platelet activation. This phenotype is comparable to the human hemolytic anemia known as paroxysmal nocturnal hemoglobinuria (PNH), in which platelet activation and thrombosis play a critical pathogenic role. It has long been suspected but not formally demonstrated that both complement and nitric oxide (NO) contribute to PNH thrombosis. Using mCd59a and mCd59b double knockout mice (mCd59ab(-/-) mice) in complement sufficient (C3(+/+)) and deficient (C3(-/-)) backgrounds, we document that mCd59ab(-/-) platelets are sensitive to complement-mediated activation and provide evidence for possible in vivo platelet activation in mCd59ab(-/-) mice. Using a combination of L-NAME (a NO-synthase inhibitor) and NOC-18 or SNAP (NO-donors), we further demonstrate that NO regulates complement-mediated activation of platelets. These results indicate that the thrombotic diathesis of PNH patients could be due to a combination of increased complement-mediated platelet activation and reduced NO-bioavailability as a consequence of hemolysis.

Stimulation of Toll-like receptor 2 in human platelets induces a thromboinflammatory response through activation of phosphoinositide 3-kinase.

Cells of the innate immune system use Toll-like receptors (TLRs) to initiate the proinflammatory response to microbial infection. Recent studies have shown acute infections are associated with a transient increase in the risk of vascular thrombotic events. Although platelets play a central role in acute thrombosis and accumulating evidence demonstrates their role in inflammation and innate immunity, investigations into the expression and functionality of platelet TLRs have been limited. In the present study, we demonstrate that human platelets express TLR2, TLR1, and TLR6. Incubation of isolated platelets with Pam(3)CSK4, a synthetic TLR2/TLR1 agonist, directly induced platelet aggregation and adhesion to collagen. These functional responses were inhibited in TLR2-deficient mice and, in human platelets, by pretreatment with TLR2-blocking antibody. Stimulation of platelet TLR2 also increased P-selectin surface expression, activation of integrin alpha(IIb)beta(3), generation of reactive oxygen species, and, in human whole blood, formation of platelet-neutrophil heterotypic aggregates. TLR2 stimulation also activated the phosphoinositide 3-kinase (PI3-K)/Akt signaling pathway in platelets, and inhibition of PI3-K significantly reduced Pam(3)CSK4-induced platelet responses. In vivo challenge with live Porphyromonas gingivalis, a Gram-negative pathogenic bacterium that uses TLR2 for innate immune signaling, also induced significant formation of platelet-neutrophil aggregates in wild-type but not TLR2-deficient mice. Together, these data provide the first demonstration that human platelets express functional TLR2 capable of recognizing bacterial components and activating the platelet thrombotic and/or inflammatory pathways. This work substantiates the role of platelets in the immune and inflammatory response and suggests a mechanism by which bacteria could directly activate platelets.

Aspirin resistance in atherosclerosis.

Clinically, aspirin resistance is defined as the failure of aspirin therapy to prevent an acute vascular thrombotic event despite regular intake of appropriate doses. In the laboratory, aspirin resistance encompasses the drug's failure to attain a particular level of platelet inhibition. From a clinical standpoint, the inability of aspirin to prevent a thrombotic event, despite appropriate cyclooxygenase-1 inhibition, implies the involvement of other factors. Evidence is emerging that aspirin resistance, as defined by residual platelet activity, merely reflects an individual's enhanced basal platelet function and suggests a hereditary component. Due to the multifactorial nature of cardiovascular disease, it is likely that a single therapy like aspirin cannot fully treat and prevent all thrombotic complications in the setting of atherosclerosis.

Neutrophil CD40 enhances platelet-mediated inflammation.

INTRODUCTION: CD40 is a transmembrane protein expressed on monocytes, macrophages, endothelial cells, and platelets. Platelets are the richest source of soluble CD40 ligand (sCD40L) and interact with monocytes and endothelial cells via CD40. While CD40 was recently reported to be present on neutrophils, the detailed mechanism of its interaction with platelets via CD40-CD40L has not been examined. MATERIALS AND METHODS: The existence of neutrophil CD40 was verified by real-time PCR and western blot. Platelet sCD40L release was measured by ELISA. Neutrophil superoxide generation was measured by chemiluminescence and confocal microscopy. The neutrophil-platelet conjugates were measured by flow cytometry. RESULTS AND CONCLUSION: The presence of neutrophils enhances stimulation-induced platelet release of sCD40L. The addition of platelets leads to an enhancement of neutrophil superoxide and reactive oxygen species (ROS) generation. The specificity of the CD40-CD40L pathway in the neutrophil-platelet interaction was confirmed by using recombinant soluble CD40L (rsCD40L) and an anti-CD40L antibody. The involvement of the PI3 kinase/Akt pathway in neutrophil superoxide production was revealed by using LY294002 in isolated neutrophils/platelets experiments, as well as during whole blood aggregation-mediated neutrophil-platelet conjugation. N-acetylcysteine, a scavenger of ROS, eliminates both neutrophil superoxide generation and sCD40L release from activated platelets. These data suggest that activated neutrophils release ROS in a PI3 kinase-dependent manner, contributing to platelet activation and further sCD40L release in a redox-controlled positive feed-back loop. In conclusion, our results define a new pathway by which platelets and neutrophils interact and modulate each other's function, and may be relevant in understanding acute thrombo-inflammatory processes.

Redox state of dipyridamole is a critical determinant for its beneficial antioxidant and antiinflammatory effects.

Dipyridamole, a well-known inhibitor of cGMP-dependent phosphodiesterase and the adenosine transporter, reportedly possesses antioxidant properties and attenuates reactive oxygen species (ROS) formation in platelet and endothelial cells. The relevance of the redox status of this compound or the mechanism for its redox-dependent effects is unknown. Oxidation of dipyridamole by horseradish peroxidase and hydrogen peroxide diminished its fluorescence and attenuated dipyridamole-mediated DPPH and ferric ferrozine reduction. Oxidation also led to elimination of dipyridamole's redox-sensitive properties, including inhibiting Cu (II)-induced LDL oxidation and ROS generation. Attenuation of activation- induced platelet release of soluble CD40 ligand (sCD40L) was diminished after dipyridamole oxidation. Dipyridamole but not oxidized dipyridamole effectively inhibited platelet adhesion to collagen-coated slides under flow conditions. By Western blot analysis, dipyridamole enhanced stimulation-induced platelet VASP phosphorylation, whereas oxidized dipyridamole caused attenuation. Using luciferase assays and nuclear translocation studies with confocal microscopy and Western blot analysis, native dipyridamole diminished TNF alpha or thrombin-induced NF kappa B activation and I kappa B alpha phosphorylation. Oxidized dipyridamole had no effect on TNFalpha-mediated NF kappa B activation. These results indicate: (1) the redox state of dipyridamole regulates its antioxidant properties; (2) dipyridamole's platelet inhibitory effects are manifested by enhanced VASP phosphorylation and platelet adhesion on collagen; and (3) dipyridamole's antioxidant effects in vascular cells are at least partially mediated via suppression of inflammatory NF kappa B signaling.

CD40-40L signaling in vascular inflammation.

Ligation of CD40 in circulating cells or in the vessel wall may promote mononuclear cell recruitment, participate in the weakening of the plaque, and contribute to thrombosis. This process appears to be redox-sensitive, but the precise signaling mechanism by which the interaction between CD40L and its receptor CD40 mediates inflammatory secretion is unclear. Our previous studies have shown that the CD40-CD40L interaction modulates release of reactive oxygen species (ROS) and the current findings demonstrate that in endothelial cells CD40L dose dependently induces intracellular CD40L and MCP1 release in a redox sensitive manner. Pharmacological inhibition of phosphatidylinositol 3-kinase and p38 MAPK as well as adenovirus-mediated inactivation of Akt and p38 MAPK inhibited CD40L effects on endothelial cells. Akt, in particular, appeared to mediate CD40L-induced CD40L synthesis and MCP1 release by endothelial cells in a redox sensitive manner via NFkappaB activation. In addition, using confocal microscopy, exogenous addition of recombinant CD40L or adenoviral mediated CD40L overexpression was found to stimulate nuclear translocation of NFkappaB, which was further augmented by Akt overexpression and inhibited by Akt inactivation. These data support a mechanism whereby redox-sensitive CD40-CD40L interactions induce activation of Akt and p38 MAPK, leading to stimulation of NFkappaB and enhanced synthesis of CD40L and MCP1. Increased CD40L and MCP1 may contribute to the adherence of CD40-positive cells, such as platelets and monocytes, to the vessel wall modulating atherothrombosis.

Compensatory mechanisms influence hemostasis in setting of eNOS deficiency.

The balance between thrombosis and hemorrhage is carefully regulated. Nitric oxide (NO) is an important mediator of these processes, as it prevents platelet adhesion to the endothelium and inhibits platelet recruitment. Although endothelial NO synthase (eNOS)-deficient mice have decreased vascular reactivity and mild hypertension, enhanced thrombosis in vivo has not been demonstrated. To determine the role of endogenous NO in hemostasis, a model of carotid arterial injury and thrombosis was performed using eNOS-deficient and wild-type mice. Paradoxically, the eNOS-deficient animals had a prolongation of time to occlusion compared with the wild-type mice (P < 0.001). Consistent with this finding, plasma markers suggesting enhanced fibrinolysis [tissue plasminogen activator (t-PA) activity and antigen and D-dimer levels] were significantly elevated in eNOS-deficient animals. Vascular tissue expression of t-PA and platelet activity levels were not altered. In endothelial cells, t-PA is stored in Weibel-Palade bodies, and exocytosis of these storage granules is inhibited by NO. Thus in the absence of NO, release of Weibel-Palade body contents (and t-PA) could be enhanced; this observation is also supported by increased von Willebrand factor levels observed in eNOS-deficient animals. In summary, although eNOS deficiency attenuates vascular reactivity and increases platelet recruitment, it is also associated with enhanced fibrinolysis due to lack of NO-dependent inhibition of Weibel-Palade body release. These processes highlight the complexity of NO-dependent regulation of vascular homeostasis. Such compensatory mechanisms may partially explain the lack of spontaneous thrombosis, minimally elevated baseline blood pressure, and normal life span that are seen in animals deficient in a pivotal regulator of vascular patency.