Platelet Rolling Research Articles

Human platelets release TGFBIp in acute myocardial infarction.

Transforming growth factor-β-induced protein (TGFBIp) is released from activated platelets and promotes pro-thrombotic complications like pulmonary embolism. The role of TGFBIp in acute coronary syndrome, especially with a focus on platelets, has not been investigated so far. Using ELISA and immunoblotting, we demonstrate platelet TGFBIp release in patients with myocardial infarction (MI). We investigated TGFBIp-induced platelet adhesion and rolling by flow chamber and chemotactic effects of TGFBIp in transwell experiments. Immunochemistry staining of arterial vessels detected TGFBIp and the platelet-specific protein GPVI in the vessel wall.We demonstrate for the first time that platelet TGFBIp release is significantly increased in MI and correlates with the severity of acute coronary syndromes (STEMI, NSTEMI). After activation with TRAP, platelets release TGFBIp and TGFBIp itself activates platelets. Under flow, TGFBIp-mediated platelet rolling and adherence similarly to collagen. TGFBIp significantly increased platelet transmigration and we demonstrate TGFBIp deposits in the wall of human arteries. In this study, we add novel aspects to the role of TGFBIp in acute coronary syndrome by demonstrating that TGFBIp is partially released from platelets during MI and has activating, pro-adhesive and pro-migratory effects on platelets that could contribute to the disease development of coronary vascular inflammation and MI.

Role of Platelets in the Pathogenicity of Atherosclerosis and Thrombosis in Coronary Heart Diseases

Atherosclerosis is a chronic inflammatory process that results in coronary artery disease, peripheral artery disease and in many cases of stroke. It is a disease that involves multiple inflammatory cytokine which is regarded as the primary underlying cause of cardiovascular diseases (CVD). CVD is the leading cause of death in the developed and developing countries like Nigeria. From pathological perspective, the chronic inflammatory condition of atherosclerosis occurs due to interplay between platelets, monocytes, macrophages. Physiologically platelets play a significant role in coagulation and repair of endothelial injury. Pathologically, studies have shown that activated platelets release multiple inflammatory cytokines and chemokines that serve as positive mediators of atherosclerosis. This chemokine is (RANTES, P-selectin and PF-4). Activated platelet release p-selectin that mediate platelet adhesion and rolling to injured endothelial cell, RANTES trigger the recruitment of monocytes into the sub- endothelium and PF4 promote the differentiation of monocytes into macrophages in the intimal layer of the endothelium which engulf ox-LDL to form FOAM cells. Thus, the aim of this review is to understand and describe the role of activated platelets in atherosclerosis as well as therapeutic target of these platelet inflammatory chemokines which is the major mediator of atherosclerosis in human.

Inhibition of Shear-Induced Platelet Aggregation by Xueshuantong via Targeting Piezo1 Channel-Mediated Ca2+ Signaling Pathway.

XueShuanTong (XST) comprising therapeutically active ginsenosides, a lyophilized extract of Panax notoginseng roots, is extensively used in traditional Chinese medicine to treat ischemic heart and cerebrovascular diseases. Our recent study shows that treatment with XST inhibits shear-induced thrombosis formation but the underlying mechanism remained unclear. This study aimed to investigate the hypothesis that XST inhibited shear-induced platelet aggregation via targeting the mechanosensitive Ca2+-permeable Piezo1 channel by performing platelet aggregation assay, Ca2+ imaging and Western blotting analysis. Exposure to shear at physiologically (1,000–2000 s−1) and pathologically related rates (4,000–6,000 s−1) induced platelet aggregation that was inhibited by treatment with GsMTx-4. Exposure to shear evoked robust Ca2+ responses in platelets that were inhibited by treatment with GsMTx-4 and conversely enhanced by treatment with Yoda1. Treatment with XST at a clinically relevant concentration (0.15 g L−1) potently inhibited shear-induced Ca2+ responses and platelet aggregation, without altering vWF-mediated platelet adhesion and rolling. Exposure to shear, while resulting in no effect on the calpain-2 expression in platelets, induced calpain-2-mediated cleavage of talin1 protein, which is known to be critical for platelet activation. Shear-induced activation of calpain-2 and cleavage of talin1 were attenuated by treatment with XST. Taken together, our results suggest that XST inhibits shear-induced platelet aggregation via targeting the Piezo1 channel to prevent Piezo1-mediated Ca2+ signaling and downstream calpain-2 and talin1 signal pathway, thus providing novel insights into the mechanism of the therapeutic action of XST on platelet aggregation and thrombosis formation.

Platelets Independently Recruit into Asthmatic Lungs and Models of Allergic Inflammation via CCR3.

Platelet activation and pulmonary recruitment occur in patients with asthma and in animal models of allergic asthma, in which leukocyte infiltration, airway remodeling, and hyperresponsiveness are suppressed by experimental platelet depletion. These observations suggest the importance of platelets to various characteristics of allergic disease, but the mechanisms of platelet migration and location are not understood. The aim of this study was to assess the mechanism of platelet recruitment to extravascular compartments of lungs from patients with asthma and after allergen challenge in mice sensitized to house dust mite (HDM) extract (contains the DerP1 [Dermatophagoides pteronyssinus extract peptidase 1] allergen); in addition, we assessed the role of chemokines in this process. Lung sections were immunohistochemically stained for CD42b+ platelets. Intravital microscopy in allergic mice was used to visualize platelets tagged with an anti–mouse CD49b-PE (phycoerythrin) antibody. Platelet–endothelial interactions were measured in response to HDM (DerP1) exposure in the presence of antagonists to CCR3, CCR4, and CXCR4. Extravascular CD42b+ platelets were detected in the epithelium and submucosa in bronchial biopsy specimens taken from subjects with steroid-naive mild asthma. Platelets were significantly raised in the lung parenchyma from patients with fatal asthma compared with postmortem control-lung tissue. Furthermore, in DerP1-sensitized mice, subsequent HDM exposure induced endothelial rolling, endothelial adhesion, and recruitment of platelets into airway walls, compared with sham-sensitized mice, via a CCR3-dependent mechanism in the absence of aggregation or interactions with leukocytes. Localization of singular, nonaggregated platelets occurs in lungs of patients with asthma. In allergic mice, platelet recruitment occurs via recognized vascular adhesive and migratory events, independently of leukocytes via a CCR3-dependent mechanism.

Aquaporin-1: a major modulator of aging-mediated platelet/endothelial dysfunction in atherothrombosis

Abstract Background Aging is associated with development of cardiovascular diseases, including atherothrombosis. Aquaporin-1 (AQP1) is a water channel that also transports hydrogen peroxide (H2O2). Regarding to the role of oxidative stress in atherothrombosis, we hypothesized that AQP1 modulates aging-associated platelet/endothelial dysfunction. Methods Human aortic endothelial cells (HAEC) from passages 5 (young) to 15 (senescent/old) were subjected to fluorescent immunocytochemistry to detect AQP1 protein expression and subcellular localization. The cells were also probed for AQP1 and the phospho/total proteins (AMPK, acetyl-coA-carboxylase (ACC), caveolin-1 and eNOS) for studying the signal transduction by immunoblotting. The endothelial cells were transfected with constructs containing H2O2 biosensor HyPer targeted to cell nucleus or cytosol followed by fluorescence imaging. The transcriptional levels of pro-inflammatory/pro-atherogenic vs. anti-inflammatory/atheroprotective genes in the cells were assessed by qRT-PCR. Human blood samples were taken and treated with or without AQP1 inhibitor (Bacopaside II, 10 μM) to examine the platelet adhesion and rolling velocity on vWF under high shear flow (100 dyn/cm2). Also, platelet aggregation in response to collagen (2 μM), ADP (1 μM) and TRAP (1 μM) were recorded. Results First, the senescence of HAEC was adjusted by a significant increase in β-galactosidase activity from passage 5 to 15. AQP1 immunofluorescence showed a remarkable increase in the young (P.5) compared to senescent (P.15) cells. Immunoblot analyses showed that aging leads to significant increases in AQP1 intensity and phosphorylation of caveolin-1 (Tyr14) and ACC (Ser79), along with decreases in phosphorylation of eNOS (Ser1177) and AMPK (Thr172) (p<0.01, n>6). Fluorescence imaging documented a robust H2O2 production in the senescent endothelial cell cytosol, but not nucleus, and activated TNF-α gene, whereas the transcription of hemoxygenase-1 gene enhanced in the young cells (p<0.01). AQP1 inhibition reduced platelet adhesion and thrombus formation, and elevated platelet rolling velocity on vWF under shear flow (p<0.01). Also, a decrease was found in platelet aggregation in response to AQP1 inhibition (p<0.05). Conclusion These studies, for the first time, demonstrate that aging induces AQP1 expression in endothelial cells and platelets, and modulates the dephosphorylation of AMPK/eNOS. These may lead to platelet/endothelial dysfunction and production of pro-coagulant/pro-inflammatory factors via ACC activation. Therefore, AQP1 inhibition could potentially be exploited as a therapeutic strategy for improving age-related atherothrombosis. Figure 1. Aging increases AQP1 expression in HAEC that leads to dephosphorylation of AMPK and eNOS. AQP inhibition also improves platelet function. *p<0.05, **p<0.01 compared to control. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Swiss National Science Foundation (SNSF)

Myeloperoxidase instigates proinflammatory responses in a cecal ligation and puncture rat model of sepsis.

Myeloperoxidase (MPO)-derived hypochlorous (HOCl) reacts with membrane plasmalogens to yield α-chlorofatty aldehydes such as 2-chlorofatty aldehyde (2-ClFALD) and its metabolite 2-chlorofatty acid (2-ClFA). Recent studies showed that 2-ClFALD and 2-ClFA serve as mediators of the inflammatory responses to sepsis by as yet unknown mechanisms. Since no scavenger for chlorinated lipids is available and on the basis of the well-established role of the MPO/HOCl/chlorinated lipid axis in inflammatory responses, we hypothesized that treatment with MPO inhibitors (N-acetyl lysyltyrosylcysteine amide or 4-aminobenzoic acid hydrazide) would inhibit inflammation and proinflammatory mediator expression induced by cecal ligation and puncture (CLP). We used intravital microscopy to quantify in vivo inflammatory responses in Sham and CLP rats with or without MPO inhibition. Small intestines, mesenteries, and lungs were collected to assess changes in MPO-positive staining and lung injury, respectively, as well as free 2-ClFA and proinflammatory mediators levels. CLP caused neutrophil infiltration, 2-ClFA generation, acute lung injury, leukocyte-/platelet-endothelium interactions, mast cell activation (MCA), plasminogen activator inhibitor-1 (PAI-1) production, and the expression of several cytokines, chemokines, and vascular endothelial growth factor, changes that were reduced by MPO inhibition. Pretreatment with a PAI-1 inhibitor or MC stabilizer prevented CLP-induced leukocyte-endothelium interactions and MCA, and abrogated exogenous 2-ClFALD-induced inflammatory responses. Thus, we provide evidence that MPO instigates these inflammatory changes in CLP and that chlorinated lipids may serve as a mechanistic link between the enzymatic activity of MPO and PAI-1- and mast cell-dependent adhesive interactions, providing a rationale for new therapeutic interventions in sepsis.NEW & NOTEWORTHY Using two distinct myeloperoxidase (MPO) inhibitors, we show for the first time that MPO plays an important role in producing increases in free 2-chlorofatty aldehyde (2-ClFALD)-a powerful proinflammatory chlorinated lipid in plasma and intestine-a number of cytokines and other inflammatory mediators, leukocyte and platelet rolling and adhesion in postcapillary venules, and lung injury in a cecal ligation and puncture model of sepsis. In addition, the use of a plasminogen activator inhibitor-1 (PAI-1) inhibitor or a mast cell stabilizer prevented inflammatory responses in CLP-induced sepsis. PAI-1 inhibition also prevented the proinflammatory responses to exogenous 2-ClFALD superfusion. Thus, our study provides some of the first evidence that MPO-derived free 2-ClFA plays an important role in CLP-induced sepsis by a PAI-1- and mast cell-dependent mechanism.

Complement C1q Enhances Primary Hemostasis.

The cross-talk between the inflammatory complement system and hemostasis is becoming increasingly recognized. The interaction between complement C1q, initiation molecule of the classical pathway, and von Willebrand factor (vWF), initiator molecule of primary hemostasis, has been shown to induce platelet rolling and adhesion in vitro. As vWF disorders result in prolonged bleeding, a lack of C1q as binding partner for vWF might also lead to an impaired hemostasis. Therefore, this study aimed to investigate the in vivo relevance of C1q-dependent binding of vWF in hemostasis. For this purpose, we analyzed parameters of primary and secondary hemostasis and performed bleeding experiments in wild type (WT) and C1q-deficient (C1qa−/−) mice, with reconstitution experiments of C1q in the latter. Bleeding tendency was examined by quantification of bleeding time and blood loss. First, we found that complete blood counts and plasma vWF levels do not differ between C1qa−/− mice and WT mice. Moreover, platelet aggregation tests indicated that the platelets of both strains of mice are functional. Second, while the prothrombin time was comparable between both groups, the activated partial thromboplastin time was shorter in C1qa−/− mice. In contrast, tail bleeding times of C1qa−/− mice were prolonged accompanied by an increased blood loss. Upon reconstitution of C1qa−/− mice with C1q, parameters of increased bleeding could be reversed. In conclusion, our data indicate that C1q, a molecule of the first-line of immune defense, actively participates in primary hemostasis by promoting arrest of bleeding. This observation might be of relevance for the understanding of thromboembolic complications in inflammatory disorders, where excess of C1q deposition is observed.

Cleavage by MMP‐13 renders VWF unable to bind to collagen but increases its platelet reactivity

BackgroundAtherosclerotic plaque rupture and subsequent thrombosis underpin thrombotic syndromes. Under inflammatory conditions in the unstable plaque, perturbed endothelial cells secrete von Willebrand Factor (VWF) which, via its interaction with GpIbα, enables platelet rolling across and adherence to the damaged endothelium. Following plaque rupture, VWF and platelets are exposed to subendothelial collagen, which supports stable platelet adhesion, activation, and aggregation. Plaque‐derived matrix metalloproteinase (MMP)‐13 is also released into the surrounding lumen where it may interact with VWF, collagen, and platelets. ObjectivesWe sought to discover whether MMP‐13 can cleave VWF and whether this might regulate its interaction with both collagen and platelets. MethodsWe have used platelet adhesion assays and whole blood flow experiments to assess the effects of VWF cleavage by MMP‐13 on platelet adhesion and thrombus formation. ResultsUnlike the shear‐dependent cleavage of VWF by a disintegrin and metalloprotease with thrombospondin motif member 13 (ADAMTS13), MMP‐13 is able to cleave VWF under static conditions. Following cleavage by MMP‐13, immobilized VWF cannot bind to collagen but interacts more strongly with platelets, supporting slower platelet rolling in whole blood under shear. Compared with intact VWF, the interaction of cleaved VWF with platelets results in greater GpIbα upregulation and P‐selectin expression, and the thrombi formed on cleaved VWF–collagen co‐coatings are larger and more contractile than platelet aggregates on intact VWF‐collagen co‐coatings or on collagen alone. ConclusionsOur data suggest a VWF‐mediated role for MMP‐13 in the recruitment of platelets to the site of vascular injury and may provide new insights into the association of MMP‐13 in atherothrombotic and stroke pathologies.

Rolling or Two-Stage Aggregation of Platelets on the Surface of Thin Ceramic Coatings under in Vitro Simulated Blood Flow Conditions.

The process of modern cardiovascular device fabrication should always be associated with an investigation of how surface properties modulate its hemocompatibility through plasma protein adsorption as well as blood morphotic element activation and adhesion. In this work, a package of novel assays was used to correlate the physicochemical properties of thin ceramic coatings with hemocompatibility under dynamic conditions. Different variants of carbon-based films were prepared on polymer substrates using the magnetron sputtering method. The microstructural, mechanical, and surface physicochemical tests were performed to characterize the coatings, followed by investigation of whole human blood quality changes under blood flow conditions using the "Impact R" test, tubes' tester, and radial flow chamber assay. The applied methodology allowed us to determine that aggregate formation on hydrophobic and hydrophilic carbon-based coatings may follow one of the two different mechanisms dependent on the type and conformational changes of adsorbed blood plasma proteins.

The key events of thrombus formation: platelet adhesion and aggregation.

Thrombus formation is a complex, dynamic and multistep process, involving biochemical reactions, mechanical stimulation, hemodynamics, and so on. In this study, we concentrate on its two crucial steps: (i) platelets adhered to a vessel wall, or simply platelet adhesion, and (ii) platelets clumping and arrested to the adherent platelets, named platelet aggregation. We report the first direct simulation of three modes of platelet adhesion, detachment, rolling adhesion and firm adhesion, as well as the formation, disintegration, arrestment and consolidation of platelet plugs. The results show that the bond dissociation in the detachment mode is mainly attributed to a high probability of rupturing bonds, such that any existing bond can be quickly ruptured and all bonds would be completely broken. In the rolling adhesion, however, it is mainly attributed to the strong traction from the shear flow or erythrocytes, causing that the bonds are ruptured at the trailing edge of the platelet. The erythrocytes play an important role in platelet activities, such as the formation, disintegration, arrestment and consolidation of platelet plugs. They exert an aggregate force on platelets, a repulsion at a near distance but an attraction at a far distance to the platelets. This aggregate force can promote platelets to form a plug and/or bring along a part of a platelet plug causing its disintegration. It also greatly influences the arrestment and consolidation of platelet plugs, together with the adhesive force from the thrombus.

Glycoprotein Ib clustering in platelets can be inhibited by α-linolenic acid as revealed by cryo-electron tomography.

Platelet adhesion to the sub-endothelial matrix and damaged endothelium occurs through a multi-step process mediated in the initial phase by glycoprotein Ib binding to von Willebrand factor (vWF), which leads to the subsequent formation of a platelet plug. The plant-derived ω-3 fatty acid α-linolenic acid is an abundant alternative to fish-derived n-3 fatty acids and has anti-inflammatory and antithrombotic properties. In this study, we investigated the impact of α-linolenic acid on human platelet binding to vWF under high-shear flow conditions (mimicking blood flow in stenosed arteries). Pre-incubation of fresh human blood from healthy donors with α-linolenic acid at dietary relevant concentrations reduced platelet binding and rolling on vWF-coated microchannels at a shear rate of 100 dyn/cm2. Depletion of membrane cholesterol by incubation of platelet-rich plasma with methyl-β cyclodextrin abrogated platelet rolling on vWF. Analysis of glycoprotein Ib by applying cryo-electron tomography to intact platelets revealed local clusters of glycoprotein Ib complexes upon exposure to shear force: the formation of these complexes could be prevented by treatment with α-linolenic acid. This study provides novel findings on the rapid local rearrangement of glycoprotein Ib complexes in response to high-shear flow and highlights the mechanism of in vitro inhibition of platelet binding to and rolling on vWF by α-linolenic acid.

Experimental and numerical study of platelets rolling on a von Willebrand factor-coated surface

Blood platelets circulate in the blood and adhere to wounded vessels to initiate coagulation and healing. The first step of this process is the capture of flowing platelets by adhesive molecules located at the wounded vessel wall. In this article, we study the transport of fixed blood platelets in a microfluidic channel coated with von Willebrand factor (vWF), a large multimeric protein expressed by endothelial cells in the vicinity of wounds. We measure the number of platelets adsorbed at the channel surface as a function of both time and space. Experimental results are compared with a new transport model. We show that transverse diffusion is an important feature of our model, while the rolling behaviour of the bounded platelets can be neglected.

Downregulation of CX3CR1 ameliorates experimental colitis: evidence for CX3CL1-CX3CR1-mediated immune cell recruitment.

Inflammatory conditions like inflammatory bowel diseases (IBD) are characterized by increased immune cell infiltration. The chemokine ligand CX3CL1 and its receptor CX3CR1 have been shown to be involved in leukocyte adhesion, transendothelial recruitment, and chemotaxis. Therefore, the objective of this study was to describe CX3CL1-CX3CR1-mediated signaling in the induction of immune cell recruitment during experimental murine colitis. Acute colitis was induced by dextran sodium sulfate (DSS), and sepsis was induced by injection of lipopolysaccharide (LPS). Serum concentrations of CX3CR1 and CX3CL1 were measured by ELISA. Wild-type and CX3CR1-/- mice were challenged with DSS, and on day 6, intravital microscopy was performed to monitor colonic leukocyte and platelet recruitment. Intestinal inflammation was assessed by disease activity, histopathology, and neutrophil infiltration. CX3CR1 was upregulated in DSS colitis and LPS-induced sepsis. CX3CR1-/- mice were protected from disease severity and intestinal injury in DSS colitis, and CX3CR1 deficiency resulted in reduced rolling of leukocytes and platelets. In the present study, we provide evidence for a crucial role of CX3CL1-CX3CR1 in experimental colitis, in particular for intestinal leukocyte recruitment during murine colitis. Our findings suggest that CX3CR1 blockade represents a potential therapeutic strategy for treatment of IBD.

Von Willebrand Factor Interacts with Surface-Bound C1q and Induces Platelet Rolling.

Premature atherosclerosis and thrombotic complications are major causes of morbidity and mortality in patients with systemic lupus erythematosus (SLE). However, the high incidence of these complications cannot be explained by traditional risk factors alone, suggesting direct effects of an activated immune system on hemostasis. The unexpected nucleotide sequence homology between SLE patient-derived autoantibodies against complement C1q (Fab anti-C1q) and von Willebrand factor (VWF) led us to investigate a potential interaction between the complement and hemostatic systems on the level of initiating molecules. VWF was found to bind to surface-bound C1q under static conditions. The binding could specifically be inhibited by Fab anti-C1q and C1q-derived peptides. Under shear stress the C1q-VWF interaction was enhanced, resembling the binding of VWF to collagen I. Additionally, we could show that C1q-VWF complexes induced platelet rolling and firm adhesion. Furthermore, we observed VWF binding to C1q-positive apoptotic microparticles and cholesterol crystals, as well as increased VWF deposition in C1q-positive glomeruli of SLE patients compared with control nephropathy. We show, to our knowledge for the first time, binding of VWF to C1q and thus a direct interaction between starter molecules of hemostasis and the classical pathway of complement. This direct interaction might contribute to the pathogenic mechanisms in complement-mediated, inflammatory diseases.

Glutamate Receptor Interacting Protein 1 Mediates Platelet Adhesion and Thrombus Formation.

Thrombosis-associated pathologies, such as myocardial infarction and stroke, are major causes of morbidity and mortality worldwide. Because platelets are necessary for hemostasis and thrombosis, platelet directed therapies must balance inhibiting platelet function with bleeding risk. Glutamate receptor interacting protein 1 (GRIP1) is a large scaffolding protein that localizes and organizes interacting proteins in other cells, such as neurons. We have investigated the role of GRIP1 in platelet function to determine its role as a molecular scaffold in thrombus formation. Platelet-specific GRIP1-/- mice were used to determine the role of GRIP1 in platelets. GRIP1-/- mice had normal platelet counts, but a prolonged bleeding time and delayed thrombus formation in a FeCl3-induced vessel injury model. In vitro stimulation of WT and GRIP1-/- platelets with multiple agonists showed no difference in platelet activation. However, in vivo platelet rolling velocity after endothelial stimulation was significantly greater in GRIP1-/- platelets compared to WT platelets, indicating a potential platelet adhesion defect. Mass spectrometry analysis of GRIP1 platelet immunoprecipitation revealed enrichment of GRIP1 binding to GPIb-IX complex proteins. Western blots confirmed the mass spectrometry findings that GRIP1 interacts with GPIbα, GPIbβ, and 14-3-3. Additionally, in resting GRIP1-/- platelets, GPIbα and 14-3-3 have increased interaction compared to WT platelets. GRIP1 interactions with the GPIb-IX binding complex are necessary for normal platelet adhesion to a stimulated endothelium.

Hypoxia Reoxygenation Treatment Induces Platelet Hyperactivity and Relieves Calpain-1-Mediated Inhibition of Platelet Aggregation in a Mouse Model of Severe Sickle Cell Disease

Introduction: With an estimated 15 million patients and no drug that addresses its etiology, sickle cell disease (SCD) remains an area of unmet need. Vaso-occlusive pain crisis (VOPC), the hallmark of SCD, is initiated by sickle RBCs (sRBCs) recruiting leukocytes and platelets to potentiate vessel occlusion. ADP released by sRBCs is a potent activator of platelets, and sickle cell patients are known to have activated platelets in circulation both at steady state and during VOPC. However, the mechanism underlying platelet dysfunction in SCD is not fully understood. Platelet activation mediated by the protease activated receptors (PAR1 and PAR4 in humans, PAR3 and PAR4 in mice), triggers PLC-β activation resulting in calcium mobilization. The increased calcium flux leads to activation of GPIIbIIIa/aIIbb3, GP1b, and P-selectin involved in platelet aggregation, adhesion, and rolling. Prior evidence has established a role of the calcium-activated cysteine protease, calpain-1 in platelet activation. Washed platelets from calpain-1 knockout C57BL/6 mice demonstrated impaired platelet aggregation. However, due to the critical contribution of sRBCs to platelet dysfunction in SCD, whole blood (impedance) aggregometry represents a physiological assessment of platelet aggregation.Methods: Townes SCD mice (SS) were backcrossed with calpain-1 knockout (CKO) mice to generate SCD mice lacking calpain-1 (SSCKO). Humanized mice (AA) were used as controls. Using flow cytometry, we evaluated in vivo platelet activation following stimulation with ADP and Thrombin GPRP. Platelet counts were obtained via ADVIA 120 and flow cytometry. For platelet aggregation, 500 μL of blood was harvested from the vena cava of AA, SS, SSCKO, and CKO mice. Whole blood aggregation in response to PAR4 stimulation was assessed using the Roche Multiplate Analyzer. A separate group of mice were challenged with hypoxia/reoxygenation (H/R) treatment (3 hours of 10.5% O2, followed by 4 hours of 21% O2) prior to platelet aggregation testing. SCD mice are characterized by tissue infarcts suggestive of thrombus formation. To examine whether H/R treatment induces formation of fibrin thrombi, we harvested brain, lungs, heart, kidneys, liver, and spleen following blood collection, and performed histology.Results: Compared to AA, SS and SSCKO mice are thrombocytopenic. Similar to Berkeley, Townes mouse platelets are activated in vivo, demonstrated by activated GPIIbIIIa on circulating platelets. At steady state, PAR4 agonist-induced platelet aggregation is similar in AA and SS mice (64 U v. 53 U, n = 6-10/group, p = 0.3). As depicted in Fig 1., SSCKO mice show significantly reduced platelet aggregation compared to SS mice (13 U v 53 U, p<0.001, n = 6-10 per group). As expected, H/R treatment induces platelet hyperactivity in SS, but not AA mice (71 U v. 59 U, p = 0.04, n = 6/group). Interestingly, the H/R-induced platelet hyperactivity partially relieves the calpain-1-mediated inhibition of platelet aggregation in SSCKO mice at steady state (Fig 2) (53 U v. 13 U, p<0.001, n = 6-8/group). Furthermore, CKO mice do not show defective platelet aggregation at both steady state, and following H/R treatment, indicating an important role for sRBCs and adhesion in whole blood aggregation. Despite attenuated platelet aggregation, SSCKO platelets at steady state show comparable expression of activated GPIIbIIIa relative to their AA and SS counterparts, indicating that the impaired aggregation at steady state is not due to defective integrin translocation. Since the Roche Multiplate Analyzer takes platelet adhesion into account in determining aggregation, we hypothesize that defective SSCKO whole blood aggregation at steady state is due to reduced cell adhesion. Finally, while silent infarcts were detected in all tissues, fibrin thrombi were not detected, a finding consistent with studies in the Berkeley model reporting minimal thrombi formation in tissues. In summary, we report that calpain-1 is required for platelet aggregation in steady state SCD but not following H/R treatment. This is the first study of H/R-induced platelet dysfunction in the Townes model, and raises the possibility of targeting calpain-1 as a treatment for platelet hyperactivity in SCD. [Display omitted] [Display omitted] DisclosuresJakubowski:Eli Lilly and Company: Employment, Equity Ownership. Frelinger:GL Synthesis: Research Funding; Celerion: Research Funding; Megakaryon: Research Funding; Bristol-Myers Squibb: Research Funding; Sysmex: Research Funding; Eisai: Research Funding; Baxalta: Research Funding; Pfizer: Research Funding; GE Global Research: Research Funding; NIH: Research Funding.

Platelet-endothelial cell interaction in brain microvessels of angiotensin II type-2 receptor knockout mice following transient bilateral common carotid artery occlusion.

The purpose of this study was to investigate the behavior of platelets (rolling and adhesion) in cerebral microvessels of angiotensin II type-2 receptor-knockout (AT2RKO) mice after transient bilateral carotid artery occlusion using intravital fluorescence microscopy. Twenty AT2RKO mice, consisting of 11 mice in the sham group and 9 mice in the ischemia reperfusion group (reperfusion after 15min of bilateral, total carotid artery occlusion) were used in this study. The hole traversed the bone and dura mater, but arachnoid, pia mater, and cerebral parenchyma were preserved. Platelets were harvested from donor mice and stained using carboxyfluorescein diacetate succinimidyl ester. The number of platelets showing rolling and adhesion to pial vessels in AT2 deficient mice at 3 and 6h after cerebral ischemia reperfusion was significantly higher than that in the sham group (P<0.05). In addition, AT2 receptor has an inhibitory role in platelet rolling and adhesion after cerebral ischemia reperfusion.

Three-dimensional multi-scale model of deformable platelets adhesion to vessel wall in blood flow.

When a blood vessel ruptures or gets inflamed, the human body responds by rapidly forming a clot to restrict the loss of blood. Platelets aggregation at the injury site of the blood vessel occurring via platelet–platelet adhesion, tethering and rolling on the injured endothelium is a critical initial step in blood clot formation. A novel three-dimensional multi-scale model is introduced and used in this paper to simulate receptor-mediated adhesion of deformable platelets at the site of vascular injury under different shear rates of blood flow. The novelty of the model is based on a new approach of coupling submodels at three biological scales crucial for the early clot formation: novel hybrid cell membrane submodel to represent physiological elastic properties of a platelet, stochastic receptor–ligand binding submodel to describe cell adhesion kinetics and lattice Boltzmann submodel for simulating blood flow. The model implementation on the GPU cluster significantly improved simulation performance. Predictive model simulations revealed that platelet deformation, interactions between platelets in the vicinity of the vessel wall as well as the number of functional GPIbα platelet receptors played significant roles in platelet adhesion to the injury site. Variation of the number of functional GPIbα platelet receptors as well as changes of platelet stiffness can represent effects of specific drugs reducing or enhancing platelet activity. Therefore, predictive simulations can improve the search for new drug targets and help to make treatment of thrombosis patient-specific.

Simvastatin reduces leucocyte– and platelet–endothelial cell interaction in murine antigen-induced arthritis in vivo

Objectives: The use of statins in the prevention and treatment of cardiovascular diseases is well established. Their use as anti-inflammatory and immunomodulatory agents in the treatment of rheumatoid arthritis (RA) has also been investigated, with several clinical and experimental studies indicating an anti-inflammatory effect of statins for RA, but other studies showing no effect or even the opposite. The current study was designed to examine the effect of simvastatin in an in vivo murine model of arthritis using intravital microscopy.Method: We assigned four groups (n = 7, female C57Bl6 mice), two with and two without antigen-induced arthritis (AiA), from which one of the non-AiA groups and one of the AiA groups were treated with simvastatin 40 mg/kg i.p. daily for 14 consecutive days after induction of arthritis. Platelet– and leucocyte–endothelial cell interaction was assessed by measurement of rolling and adherent fluorescence-labelled platelets and leucocytes, functional capillary density (FCD) was evaluated, and knee joint diameter was determined as a clinical parameter.Results: In arthritic mice treated with simvastatin, a significant reduction in platelet– and leucocyte–endothelial cell interaction was observed in comparison to arthritic mice treated with vehicle. In addition, a significant reduction in FCD was seen in arthritic mice treated with simvastatin, along with a reduction in knee joint swelling of the AiA mice.Conclusions: Treatment of AiA mice with simvastatin showed significant reductions in platelet– and leucocyte–endothelial cell interactions, in FCD, and in the swelling of the knee joint. These results support the hypothesis of the anti-inflammatory effects of statins in the treatment of RA.

The integrin antagonist, cilengitide, is a weak inhibitor of αIIbβ3 mediated platelet activation and inhibits platelet adhesion under flow

The RGD cyclic pentapetide, cilengitide, is a selective inhibitor of αvβ3 and αvβ5 integrins and was developed for antiangiogenic therapy. Since cilengitide interacts with platelet αIIbβ3 and platelets express αv integrins, the effect of cilengitide on platelet pro-coagulative response and adhesion is of interest. Flow-based adhesion assays were performed to evaluate platelet adhesion and rolling on von Willebrand factor (vWf), on fibrinogen and on human umbilical vein endothelial cells (HUVECs). Flow cytometry was used to detect platelet activation (PAC1) and secretion (CD62P) by cilengitide and light transmission aggregometry was used to detect cilengitide-dependent platelet aggregation. Cilengitide inhibited platelet adhesion to fibrinogen at concentrations above 250 µM [which is the Cmax in human studies] and adhesion to vWf and HUVECs at higher concentrations under physiologic flow conditions. Platelet aggregation was already impaired at cilengitide concentrations >10 µM. Activation of αIIbβ3 integrin was inhibited by 250 µM cilengitide, whereas platelet secretion was unaffected by cilengitide. No evidence of cilengitide-induced platelet activation was found at all tested concentrations (0.01–1500 µM). At higher concentrations, platelet activation was inhibited, predominantly due to αIIbβ3 inhibition.