A new function for MAP4K4 inhibitors during platelet aggregation and platelet-mediated clot retraction.

Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) is implicated in type 2 diabetes mellitus, insulin tolerance, inflammation, cancer, and atherosclerosis. We found that GNE 495 and PF 06260933 (both potent and selective MAP4K4 inhibitors) regulated human platelet activation. Immunoblotting revealed human platelets express MAP4K4, and that GNE 495 and PF 06260933 inhibited collagen-, ADP-, and thrombin-induced platelet aggregation and eventually suppressed granule release, TXA2 generation, integrin αIIbß3 activation, and clot retraction. In addition, both inhibitors elevated intracellular levels of cAMP, and coincubation with GNE 495 and aspirin or dipyridamole (a phosphodiesterase inhibitor) synergistically inhibited collagen-induced platelet aggregation and TXA2 generation. Moreover, both inhibitors phosphorylated VASP (ser157), IP3 receptor, and PKA and attenuated MAPK and PI3K/Akt/GSK3ß signaling pathways. This study is the first to demonstrate that MAP4K4 inhibitors reduce thrombus formation by inhibiting platelet activation. These findings also suggest MAP4K4 be considered an emerging target protein for the treatment of thrombosis.

Characterization of Integrin αIIbß3-Mediated Outside-in Signaling by Protein Kinase Cδ in Platelets.

Engagement of integrin αIIbß3 promotes platelet-platelet interaction and stimulates outside-in signaling that amplifies activation. Protein kinase Cδ (PKCδ) is known to play an important role in platelet activation, but its role in outside-in signaling has not been established. In the present study, we determined the role of PKCδ and its signaling pathways in integrin αIIbß3-mediated outside-in signaling in platelets using PKCδ-deficient platelets. Platelet spreading to immobilized fibrinogen resulted in PKCδ phosphorylation, suggesting that αIIbß3 activation caused PKCδ activation. αIIbß3-mediated phosphorylation of Akt was significantly inhibited in PKCδ -/- platelets, indicating a role of PKCδ in outside-in signaling. αIIbß3-mediated PKCδ phosphorylation was inhibited by proline-rich tyrosine kinase 2 (Pyk2) selective inhibitor, suggesting that Pyk2 contributes to the regulation of PKCδ phosphorylation in outside-in signaling. Additionally, Src-family kinase inhibitor PP2 inhibited integrin-mediated Pyk2 and PKCδ phosphorylation. Lastly, platelet spreading was inhibited in PKCδ -/- platelets compared to the wild-type (WT) platelets, and clot retraction from PKCδ -/- platelets was markedly delayed, indicating that PKCδ is involved in the regulation of αIIbß3-dependent interactivities with cytoskeleton elements. Together, these results provide evidence that PKCδ plays an important role in outside-in signaling, which is regulated by Pyk2 in platelets.

The G-protein ßγ subunits regulate platelet function.

AIMS: G-protein coupled receptors (GPCRs) tightly regulate platelet function by interacting with various physiological agonists. An essential mediator of GPCR signaling is the G protein αßγ heterotrimers, in which the ßγ subunits are central players in downstream signaling. Herein, we investigated the role of Gßγ subunits in platelet function, hemostasis and thrombogenesis. METHODS: To achieve this goal, platelets from both mice and humans were employed in the context of a small molecule inhibitor of Gßγ, namely gallein. We used an aggregometer to examine aggregation and dense granules secretion. We also used flow cytometry for P-selectin and PAC1 to determine the impact of inhibiting Gßγ on α -granule secretion and αIIbß3 activation. Clot retraction and the platelet spreading assay were used to examine Gßγ role in outside-in platelet signaling, whereas Western blot was employed to examine its role in Akt activation. Finally, we used the bleeding time assay and the FeCl3-induced carotid-artery injury thrombosis model to determine Gßγ contribution to in vivo platelet function. RESULTS: We observed that gallein inhibits platelet aggregation and secretion in response to agonist stimulation, in both mouse and human platelets. Furthermore, gallein also exerted inhibitory effects on integrin αIIbß3 activation, clot retraction, platelet spreading and Akt activation/phosphorylation. Finally, gallein's inhibitory effects manifested in vivo, as documented by its ability to modulate physiological hemostasis and delay thrombus formation. CONCLUSION: Our findings demonstrate, for the first time, that Gßγ subunits directly regulate GPCR-dependent platelet function, in vitro and in vivo. Moreover, these data highlight Gßγ as a novel therapeutic target for managing thrombotic disorders.

Clot Retraction: A Miniaturized Hemoretractometer for Blood Clot Retraction Testing (Small 29/2016).

Whole blood coagulation testing provides valuable diagnostic information on diseases such as bleeding disorders, heart attack, deep venous thrombosis, etc. On page 3926, J. Fu and co-workers develop a miniaturized hemoretractometer to measure clot contraction upon blood coagulation with good reproducibility and robustness. This device design shows great application potential in point-of-care testing. Photo credit: David Peyer from University of Michigan.

A Miniaturized Hemoretractometer for Blood Clot Retraction Testing.

Blood coagulation is a critical hemostatic process that must be properly regulated to maintain a delicate balance between bleeding and clotting. Disorders of blood coagulation can expose patients to the risk of either bleeding disorders or thrombotic diseases. Coagulation diagnostics using whole blood is very promising for assessing the complexity of the coagulation system and for global measurements of hemostasis. Despite the clinic values that existing whole blood coagulation tests have demonstrated, these systems have significant limitations that diminish their potential for point-of-care applications. Here, recent advancements in device miniaturization using functional soft materials are leveraged to develop a miniaturized clot retraction force assay device termed mHemoRetractoMeter (mHRM). The mHRM is capable of precise measurements of dynamic clot retraction forces in real time using minute amounts of whole blood. To further demonstrate the clinical utility of the mHRM, systematic studies are conducted using the mHRM to examine the effects of assay temperature, treatments of clotting agents, and pro- and anti-coagulant drugs on clot retraction force developments of whole blood samples. The mHRM's low fabrication cost, small size, and consumption of only minute amounts of blood samples make the technology promising as a point-of-care tool for future coagulation monitoring.

Reduced clot retraction rate and altered platelet energy production in patients with asthma.

OBJECTIVE: Asthma enhances the risk of pulmonary embolism. The mechanism of this phenomenon is unclear. METHODS: We evaluated the kinetics of clot formation, clot retraction rate (CRR), clot volume at 40 min, the rate of lactate production (a marker of aerobic glycolysis in platelets in contracting clots), blood eosinophil count (EOS), nitric oxide in exhaled breath (FENO), and spirometry (FEV1) in 50 healthy controls and in 81 allergic asthmatics (41 subjects with steroid-naïve asthma and 40 with steroid-treated asthma). RESULTS: Thromboelastometry revealed that only steroid-treated asthmatics had slightly activated coagulation. Compared with healthy controls, whole asthmatics demonstrated (p < 0.05) reduced CRR, higher clot volume at 40 minutes, higher FENO, decreased FEV1, elevated EOS, and augmented lactate production in retracting clots. Reduced CRR was observed also in the absence of native plasma. In whole study population (asthmatics and healthy controls), CRR positively correlated with spirometry (rS = 0.668, p = <0.001) and negatively with FENO (rS = -0.543; p < 0.001), EOS (rS = -0.367, p < 0.002), and lactate production (rS = -0.791; p < 0.001). However, in steroid-treated asthmatics, the CRR did not correlate with FENO and EOS. In all study patients lactate production negatively correlated with FEV1 and positively with FENO. CONCLUSION: Collectively, this data is consistent with the hypothesis that, in asthmatics, reactive nitrogen species produced in the lungs may reduce platelet contractility (and CRR) through the diminution of platelet energy production. CRR inhibition would predispose asthmatics to pulmonary embolism.

Kinetics and mechanics of clot contraction are governed by the molecular and cellular composition of the blood.

Platelet-driven blood clot contraction (retraction) is thought to promote wound closure and secure hemostasis while preventing vascular occlusion. Notwithstanding its importance, clot contraction remains a poorly understood process, partially because of the lack of methodology to quantify its dynamics and requirements. We used a novel automated optical analyzer to continuously track in vitro changes in the size of contracting clots in whole blood and in variously reconstituted samples. Kinetics of contraction was complemented with dynamic rheometry to characterize the viscoelasticity of contracting clots. This combined approach enabled investigation of the coordinated mechanistic impact of platelets, including nonmuscle myosin II, red blood cells (RBCs), fibrin(ogen), factor XIIIa (FXIIIa), and thrombin on the kinetics and mechanics of the contraction process. Clot contraction is composed of 3 sequential phases, each characterized by a distinct rate constant. Thrombin, Ca(2+), the integrin αIIbß3, myosin IIa, FXIIIa cross-linking, and platelet count all promote 1 or more phases of the clot contraction process. In contrast, RBCs impair contraction and reduce elasticity, while increasing the overall contractile stress generated by the platelet-fibrin meshwork. A better understanding of the mechanisms by which blood cells, fibrin(ogen), and platelet-fibrin interactions modulate clot contraction may generate novel approaches to reveal and to manage thrombosis and hemostatic disorders.

Nucleotide-binding oligomerization domain 2 receptor is expressed in platelets and enhances platelet activation and thrombosis.

BACKGROUND: Pattern recognition receptor nucleotide-binding oligomerization domain 2 (NOD2) is well investigated in immunity, but its expression and function in platelets has never been explored. METHOD AND RESULTS: Using reverse transcription polymerase chain reaction and Western blot, we show that both human and mouse platelets express NOD2, and its agonist muramyl dipeptide induced NOD2 activation as evidenced by receptor dimerization. NOD2 activation potentiates platelet aggregation and secretion induced by low concentrations of thrombin or collagen, and clot retraction, as well. These potentiating effects of muramyl dipeptide were not seen in platelets from NOD2-deficient mice. Plasma from septic patients also potentiates platelet aggregation induced by thrombin or collagen NOD2 dependently. Using intravital microscopy, we found that muramyl dipeptide administration accelerated in vivo thrombosis in a FeCl3-injured mesenteric arteriole thrombosis mouse model. Platelet depletion and transfusion experiments confirmed that NOD2 from platelets contributes to the in vivo thrombosis in mice. NOD2 activation also accelerates platelet-dependent hemostasis. We further found that platelets express receptor-interacting protein 2, and provided evidence suggesting that mitogen activated-protein kinase and nitric oxide/soluble guanylyl cyclase/cGMP/protein kinase G pathways downstream of receptor-interacting protein mediate the role of NOD2 in platelets. Finally, muramyl dipeptide stimulates proinflammatory cytokine interleukin-1ß maturation and accumulation in human and mouse platelets NOD2 dependently. CONCLUSIONS: NOD2 is expressed in platelets and functions in platelet activation and arterial thrombosis, possibly during infection. To our knowledge, this is the first study on NOD-like receptors in platelets that link thrombotic events to inflammation.

Platelet-related fibrinolysis resistance in patients suffering from PV. Impact of clot retraction and isovolemic erythrocytapheresis.

Using patients with polycythemia vera (PV) as an experimental model, we evaluated the impact of clot retraction (CR) and architecture of the clot on fibrinolysis. We studied the kinetics of clot retraction and the fibrinolysis rate in whole blood from 48 PV patients and 48 healthy controls. Measurements were performed before and after isovolemic eryhrocytapheresis (ECP). CR was measured by optical method. Clot lysis time (CLT) and maximum clot firmness (MCF) were measured by thromboelastometry in recalcified blood supplemented with t-PA and tissue factor. Compared with healthy controls, CR rate in PV patients was higher (0.0219 vs. 0.0138; p<0.001), the clot volume smaller and MCF elevated (64 vs. 58 mm). CR rate correlated with platelet count (r=0.546; p=0.001) but not with erythrocyte concentration (r=0.192; p>0.3). Compared with healthy controls, CLT in PV patients was significantly prolonged (158 min vs. 71 min). Fibrinolysis rate inversely correlated with CR rate (r=-0.566; p<0.001); MCF (r=-0.704; p<0.001) and platelet count (r=-0.461; p<0.001). As judged by confocal microscope, in comparison to healthy controls, clots formed in blood from PV patients demonstrated booth a distinctly higher degree of crosslinking and possessed thinner fibers. Altered CR, MCF and fibrinolysis speeds were not normalized following the ECP procedure. Tirofiban (a blocker of platelet GPIIb/IIIa receptors), unlike aspirin, normalized abnormal CR and fibrinolysis in blood from PV patients. Collectively, our data indicate that in PV patients, abnormal CR may result in formation of thrombi that are more resistant to fibrinolysis. ECP and aspirin failed to normalize platelet-related fibrinolysis resistance.

Disulfide bond exchanges in integrins αIIbß3 and αvß3 are required for activation and post-ligation signaling during clot retraction.

BACKGROUND: Integrin αIIbß3 mediates platelet adhesion, aggregation and fibrin clot retraction. These processes require activation of αIIbß3 and post-ligation signaling. Disulfide bond exchanges are involved in αIIbß3 and αvß3 activation. METHODS: In order to investigate the role of integrin activation and disulfide bond exchange during αIIbß3- and αvß3-mediated clot retraction, we co-expressed in baby hamster kidney cells wild-type (WT) human αIIb and WT or mutated human ß3 that contain single or double cysteine substitutions disrupting C523-C544 or C560-C583 bonds. Flow cytometry was used to measure surface expression and activation state of the integrins. Time-course of fibrin clot retraction was examined. RESULTS: Cells expressed WT or mutated human αIIbß3 as well as chimeric hamster/human αvß3. The αIIbß3 mutants were constitutively active and the thiol blocker dithiobisnitrobenzoic acid (DTNB) did not affect their activation state. WT cells retracted the clot and addition of αvß3 inhibitors decreased the retraction rate. The active mutants and WT cells activated by anti-LIBS6 antibody retracted the clot faster than untreated WT cells, particularly in the presence of αvß3 inhibitor. DTNB substantially inhibited clot retraction by WT or double C523S/C544S mutant expressing cells, but minimally affected single C523S, C544S or C560S mutants. Anti-LIBS6-enhanced clot retraction was significantly inhibited by DTNB when added prior to anti-LIBS6. CONCLUSIONS: Both αIIbß3 and αvß3 contribute to clot retraction without prior activation of the integrins. Activation of αIIbß3, but not of αvß3 enhances clot retraction. Both αIIbß3 activation and post-ligation signaling during clot retraction require disulfide bond exchange.

Peroxynitrite may affect clot retraction in human blood through the inhibition of platelet mitochondrial energy production.

Peroxynitrite (ONOO(-)) contributes to hemostasis abnormalities associated with inflammatory states by a poorly understood mechanism. Here we show that ONOO(-) may affect clot retraction (CR), an important step in hemostasis, by reducing contractility of human platelets resulting from the inhibition of mitochondrial energy production. Reduced CR may result in thromboembolic and hemorrhage events. The results show that in human blood, in vitro, physiologically relevant ONOO(-) concentrations reduce clot retraction rate and enlarge final clot size. The stressor was more effective in reconstituted system consisting of washed platelets and fibrinogen, (IC50=25 nM) than in platelet rich plasma (IC50=75 µM) or in whole blood (IC50=120 µM), indicating that its effect depends on the number of targets. Retardation of CR by lower concentrations of ONOO(-) resulted in reduction of platelet energy production due to impairment of mitochondria but not from tyrosine nitration or inhibition of actin polymerization. In washed platelets nanomolar ONOO(-) concentrations produced a drop of the mitochondrial transmembrane potential (ΔΨm) explaining high sensitivity of CR (a large consumer of platelet energy) to stressor. Thromboelastometry measurements showed that ONOO(-) may diminish clot stability and elasticity through the reduction of platelet contractility. Our findings suggest that in humans ONOO(-)- altered platelet mitochondria represent a new link between inflammation and hemostasis.

Biphasic myosin II light chain activation during clot retraction.

Clot retraction is an essential step during primary haemostasis, thereby promoting thrombus stability and wound healing. Integrin αIIbß3 plays a critical role in clot retraction, by inducing acto-myosin interactions that allow platelet cytoskeleton reorganisation. However, the signalling pathways that lead to clot retraction are still misunderstood. In this study, we report the first data on the kinetics of myosin II light chain (MLC) phosphorylation during clot retraction. We found an early phosphorylation peak followed by a second peak. By using specific inhibitors of kinases and small G proteins, we showed that MLC kinase (MLCK), RhoA/ROCK, and Rac-1 were involved in clot retraction and in the early MLC phosphorylation peak. Only Rac-1 and actin polymerisation, controlled by outside-in signalling, were crucial to the second MLC phosphorylation peak.

Gap junctions and connexin hemichannels underpin hemostasis and thrombosis.

BACKGROUND: Connexins are a widespread family of membrane proteins that assemble into hexameric hemichannels, also known as connexons. Connexons regulate membrane permeability in individual cells or couple between adjacent cells to form gap junctions and thereby provide a pathway for regulated intercellular communication. We have examined the role of connexins in platelets, blood cells that circulate in isolation but on tissue injury adhere to each other and the vessel wall to prevent blood loss and to facilitate wound repair. METHODS AND RESULTS: We report the presence of connexins in platelets, notably connexin37, and that the formation of gap junctions within platelet thrombi is required for the control of clot retraction. Inhibition of connexin function modulated a range of platelet functional responses before platelet-platelet contact and reduced laser-induced thrombosis in vivo in mice. Deletion of the Cx37 gene (Gja4) in transgenic mice reduced platelet aggregation, fibrinogen binding, granule secretion, and clot retraction, indicating an important role for connexin37 hemichannels and gap junctions in platelet thrombus function. CONCLUSIONS: Together, these data demonstrate that platelet gap junctions and hemichannels underpin the control of hemostasis and thrombosis and represent potential therapeutic targets.

Tyrosine phosphorylated c-Cbl regulates platelet functional responses mediated by outside-in signaling.

c-Cbl protein functions as an E3 ligase and scaffolding protein, where 3 residues, Y700, Y731, and Y774, upon phosphorylation, have been shown to initiate several signaling cascades. In this study, we investigated the role of these phospho-tyrosine residues in the platelet functional responses after integrin engagement. We observed that c-Cbl Y700, Y731 and Y774 undergo phosphorylation upon platelet adhesion to immobilized fibrinogen, which was inhibited in the presence of PP2, a pan-src family kinase (SFK) inhibitor, suggesting that c-Cbl is phosphorylated downstream of SFKs. However, OXSI-2, a Syk inhibitor, significantly reduced c-Cbl phosphorylation at residues Y774 and Y700, without affecting Y731 phosphorylation. Interestingly, PP2 inhibited both platelet-spreading on fibrinogen as well as clot retraction, whereas OXSI-2 blocked only platelet-spreading, suggesting a differential role of these tyrosine residues. The physiologic role of c-Cbl and Y731 was studied using platelets from c-Cbl KO and c-Cbl(YF/YF) knock-in mice. c-Cbl KO and c-Cbl(YF/YF) platelets had a significantly reduced spreading over immobilized fibrinogen. Furthermore, clot retraction with c-Cbl KO and c-Cbl(YF/YF) platelets was drastically delayed. These results indicate that c-Cbl and particularly its phosphorylated residue Y731 plays an important role in platelet outside-in signaling contributing to platelet-spreading and clot retraction.

Distinct roles for Rap1b protein in platelet secretion and integrin αIIbß3 outside-in signaling.

Rap1b is activated by platelet agonists and plays a critical role in integrin α(IIb)ß(3) inside-out signaling and platelet aggregation. Here we show that agonist-induced Rap1b activation plays an important role in stimulating secretion of platelet granules. We also show that α(IIb)ß(3) outside-in signaling can activate Rap1b, and integrin outside-in signaling-mediated Rap1b activation is important in facilitating platelet spreading on fibrinogen and clot retraction. Rap1b-deficient platelets had diminished ATP secretion and P-selectin expression induced by thrombin or collagen. Importantly, addition of low doses of ADP and/or fibrinogen restored aggregation of Rap1b-deficient platelets. Furthermore, we found that Rap1b was activated by platelet spreading on immobilized fibrinogen, a process that was not affected by P2Y(12) or TXA(2) receptor deficiency, but was inhibited by the selective Src inhibitor PP2, the PKC inhibitor Ro-31-8220, or the calcium chelator demethyl-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis. Clot retraction was abolished, and platelet spreading on fibrinogen was diminished in Rap1b-deficient platelets compared with wild-type controls. The defects in clot retraction and spreading on fibrinogen of Rap1b-deficient platelets were not rescued by addition of MnCl(2), which elicits α(IIb)ß(3) outside-in signaling in the absence of inside-out signaling. Thus, our results reveal two different activation mechanisms of Rap1b as well as novel functions of Rap1b in platelet secretion and in integrin α(IIb)ß(3) outside-in signaling.

Talin-dependent integrin activation is required for fibrin clot retraction by platelets.

Talin functions both as a regulator of integrin affinity and as an important mechanical link between integrins and the cytoskeleton. Using genetic deletion of talin, we show for the first time that the capacity of talin to activate integrins is required for fibrin clot retraction by platelets. To further dissect which talin functions are required for this process, we tested clot retraction in platelets expressing a talin1(L325R) mutant that binds to integrins, but exhibits impaired integrin activation ascribable to disruption of the interaction between talin and the membrane-proximal region (MPR) in the ß-integrin cytoplasmic domain. Talin-deficient and talin1(L325R) platelets were defective in retracting fibrin clots. However, the defect in clot retraction in talin1(L325R) platelets, but not talin-deficient platelets, was rescued by extrinsically activating integrins with manganese, thereby proving that integrin activation is required and showing that talin1(L325R) can form functional links to the actin cytoskeleton.

Platelet quality after washing: the effect of storage time before washing.

BACKGROUND: Patients who have experienced anaphylactic transfusion reactions receive washed platelet (PLT) concentrates (PCs) where the plasma has been substituted with a PLT additive solution. This study compares the in vitro quality of PCs washed at the beginning of the storage period (Day 1) to PCs washed at the end of storage (Day 7). STUDY DESIGN AND METHODS: PLTs were prepared by the buffy coat procedure. Two concentrates were pooled and then split to obtain an identical pair of PCs. One of the PCs was washed with T-Sol on Day 1 and the other on Day 7 of storage. Swirling, blood gases, and metabolic variables were analyzed before washing. Analyses of surface expression of CD62P and coagulation by free oscillation rheometry (FOR) were performed before and after washing. RESULTS: pH was acceptable in all PCs. Washing on Days 1 and 7 increased the CD62P surface expression. The FOR variables clotting time and clot retraction were not influenced by washing on either day. Washing resulted in a decrease in the number of PLTs and the decrease was larger on Day 7 compared to Day 1. CONCLUSIONS: PLTs washed on Days 1 and 7 of storage are effected by washing in a similar manner. However, a larger loss of PLTs occurred during washing on Day 7.

Phosphoinositide 3-kinase p110 beta regulates integrin alpha IIb beta 3 avidity and the cellular transmission of contractile forces.

Phosphoinositide (PI) 3-kinase (PI3K) signaling processes play an important role in regulating the adhesive function of integrin alpha(IIb)beta(3), necessary for platelet spreading and sustained platelet aggregation. PI3K inhibitors are effective at reducing platelet aggregation and thrombus formation in vivo and as a consequence are currently being evaluated as novel antithrombotic agents. PI3K regulation of integrin alpha(IIb)beta(3) activation (affinity modulation) primarily occurs downstream of G(i)-coupled and tyrosine kinase-linked receptors linked to the activation of Rap1b, AKT, and phospholipase C. In the present study, we demonstrate an important role for PI3Ks in regulating the avidity (strength of adhesion) of high affinity integrin alpha(IIb)beta(3) bonds, necessary for the cellular transmission of contractile forces. Using knock-out mouse models and isoform-selective PI3K inhibitors, we demonstrate that the Type Ia p110 beta isoform plays a major role in regulating thrombin-stimulated fibrin clot retraction in vitro. Reduced clot retraction induced by PI3K inhibitors was not associated with defects in integrin alpha(IIb)beta(3) activation, actin polymerization, or actomyosin contractility but was associated with a defect in integrin alpha(IIb)beta(3) association with the contractile cytoskeleton. Analysis of integrin alpha(IIb)beta(3) adhesion contacts using total internal reflection fluorescence microscopy revealed an important role for PI3Ks in regulating the stability of high affinity integrin alpha(IIb)beta(3) bonds. These studies demonstrate an important role for PI3K p110 beta in regulating the avidity of high affinity integrin alpha(IIb)beta(3) receptors, necessary for the cellular transmission of contractile forces. These findings may provide new insight into the potential antithrombotic properties of PI3K p110 beta inhibitors.

Differential inflammatory activity across human abdominal aortic aneurysms reveals neutrophil-derived leukotriene B4 as a major chemotactic factor released from the intraluminal thrombus.

Development and progression of acquired abdominal aortic aneurysms (AAAs) have been associated with different inflammatory mediators. The aim of the present study was to elucidate the topology and the potential mechanisms linking the leukotriene pathway to human AAAs. Human aneurysmal lesions were obtained from 24 patients undergoing surgery, and the intraluminal thrombus was separated from the vascular wall. Histological examination revealed major expression of the leukotriene-producing enzymes 5-lipoxygenase and LTA(4) hydrolase, as well as the two receptors for leukotriene B(4) (BLT1R and BLT2R), corresponding to neutrophils in the luminal part of the thrombus. In contrast, in the vascular wall, the leukotriene pathway mainly localized in macrophage-rich adventitial areas. Furthermore, conditioned media of the intraluminal thrombus contained significantly higher concentrations of leukotriene B(4) than that derived from the vascular wall, which were significantly correlated to other neutrophil-derived mediators, such as elastase/alpha(1)-antitrypsin complexes, myeloperoxidase, and MMP9/NGAL complexes. Finally, the neutrophil-chemotactic activity of the conditioned media from the intraluminal thrombus exhibited major inhibition by antagonists of the leukotriene B(4) receptors. Taken together, these results indicate neutrophil-derived leukotriene B(4) as a major neutrophil chemotactic factor released from the intraluminal thrombus of human AAAs and suggest that targeting BLT receptors may represent a potential medical therapeutic strategy in the prevention of AAA progression in humans.

Identification of a fibrin-independent platelet contractile mechanism regulating primary hemostasis and thrombus growth.

A fundamental property of platelets is their ability to transmit cytoskeletal contractile forces to extracellular matrices. While the importance of the platelet contractile mechanism in regulating fibrin clot retraction is well established, its role in regulating the primary hemostatic response, independent of blood coagulation, remains ill defined. Real-time analysis of platelet adhesion and aggregation on a collagen substrate revealed a prominent contractile phase during thrombus development, associated with a 30% to 40% reduction in thrombus volume. Thrombus contraction developed independent of thrombin and fibrin and resulted in the tight packing of aggregated platelets. Inhibition of the platelet contractile mechanism, with the myosin IIA inhibitor blebbistatin or through Rho kinase antagonism, markedly inhibited thrombus contraction, preventing the tight packing of aggregated platelets and undermining thrombus stability in vitro. Using a new intravital hemostatic model, we demonstrate that the platelet contractile mechanism is critical for maintaining the integrity of the primary hemostatic plug, independent of thrombin and fibrin generation. These studies demonstrate an important role for the platelet contractile mechanism in regulating primary hemostasis and thrombus growth. Furthermore, they provide new insight into the underlying bleeding diathesis associated with platelet contractility defects.