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

Objective: Coagulation factor XI (FXI) has been shown to contribute to thrombus formation on collagen or tissue factor (TF)-coated surfaces in vitro and in vivo by enhancing thrombin generation. Whether the role of the intrinsic pathway of coagulation is restricted to the local site of thrombus formation is unknown. This study was designed to determine whether FXI could promote both proximal and distal platelet activation and aggregate formation in the bloodstream. Approach and Results: Pharmacological blockade of FXI activation or thrombin activity in blood did not affect local platelet adhesion, yet reduced local platelet aggregation, thrombin localization and fibrin formation on immobilized collagen and TF under shear flow, ex vivo . Downstream of the thrombus formed on immobilized collagen or collagen and 10 pM TF, platelet CD62P expression and microaggregate formation and progressive platelet consumption were significantly reduced in the presence of FXI-function blocking antibodies or a thrombin inhibitor in a shear rate- and time-dependent manner. In a non-human primate model of thrombus formation, we found that inhibition of FXI reduced single platelet consumption in the bloodstream distal to a site of thrombus formation. Conclusions: This study demonstrates that the FXI-thrombin axis contributes to distal platelet activation and procoagulant microaggregate formation in the blood flow downstream of the site of thrombus formation. Our data highlights FXI as a novel therapeutic target for inhibiting distal platelet activation without affecting proximal platelet adhesion.

Download Full-text

Collagen Receptor Signaling in Platelets and Megakaryocytes

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615855 ◽

1999 ◽

Vol 82 (08) ◽

pp. 365-376 ◽

Cited By ~ 75

Author(s):

Steve Watson

Keyword(s):

Platelet Activation ◽

Platelet Adhesion ◽

Collagen Fibers ◽

Severe Bleeding ◽

Platelet Surface ◽

Collagen Receptors ◽

Intracellular Signals ◽

Von Willebrand ◽

Surface Glycoproteins

IntroductionThe extracellular matrix protein, collagen, plays a primary role in hemostasis. Collagen fibers provide an important site for adhesion of platelets to the exposed subendothelium, trapping them at the site of vascular damage and enabling the formation of a monolayer of cells over the damaged area. Collagen fibers also stimulate platelet activation, leading to inside-out regulation of the integrin glycoprotein (GP) IIb-IIIa (also known as αIIbβ3), secretion from dense and α granules, generation of thromboxanes, and expression of procoagulant activity, all of which support the hemostatic process. The role of collagen in supporting platelet adhesion to the subendothelium is mediated through indirect and direct interactions. The indirect interaction is mediated through von Willebrand factor (vWF), which binds to the GP Ib-IX-V complex on the platelet surface.1-3 The interaction with vWF is critical for platelet adhesion at medium to high rates of flow because of the fast rate of association between vWF and GP Ib-IX. The importance of this interaction is demonstrated by the severe bleeding problems experienced by individuals with functional impairment of vWF (von Willebrand disease) or GP Ib-IX (Bernard-Soulier syndrome). At low rates of flow, collagen fibers are able to support adhesion in the absence of vWF through a direct interaction with a number of platelet surface glycoproteins i.e. collagen receptors,4,5 this also serves to support vWF-dependent adhesion at higher rates of flow by preventing dissociation. Crosslinking of platelet surface glycoproteins by collagen also generates intracellular signals, leading to platelet activation.The number of proteins on the platelet surface proposed to be collagen receptors is approaching double figures, but it is generally accepted that the integrin GP Ia-IIa (also known as α2β1) and glycoprotein VI (GP VI) are among the most important of these, playing critical roles in adhesion and activation, respectively6 (Fig. 1). This is illustrated by the mild bleeding problems of patients with a low level of expression or the presence of autoantibodies to GP Ia-IIa and the spontaneous, severe bleeding episodes that are occasionally seen in patients whose platelets are deficient in GP VI.6 There is evidence, however, that other collagen receptors have supporting roles in adhesion and activation. For example, GP VI supports platelet adhesion to collagen7 and GP IV, also known as CD36, may also play a similar role.8 The role of the recently cloned collagen receptor p65 in adhesion is not known. Evidence that the interaction of collagen with receptors, such as GPIV and p65, is of less importance than for interactions with GP Ia-IIa, and GP VI is provided by the absence of individuals with bleeding problems caused by deficiencies in these proteins. This is illustrated most clearly for GP IV, which is absent in 3% to 5 % of the Japanese population, and yet such individuals display no major vascular problems.Due to the large number of glycoproteins that bind collagen on the platelet surface, it has been difficult to gain a full understanding of the role of individual collagen receptors in adhesion and activation responses. This is complicated further by the interactions between vWF and GP Ib-IX-V, vWF or fibrinogen to activated GP IIb-IIIa especially as both glycoprotein receptors generate intracellular signals. The relative importance of individual collagen receptors in adhesion also varies with the rate of flow and between collagen types. A full discussion of platelet adhesion to collagen is beyond the scope of this article, and the reader is referred to a number of excellent recent reviews for further information.4-6,9,10 The present chapter focuses on the signaling events generated by the activation (or more correctly crosslinking) of platelet surface glycoproteins by collagen and the implications that this has for platelet activation under normal and diseased conditions.

Download Full-text

Platelets selectively enhance lymphocyte adhesion on subendothelial matrix under arterial flow conditions

Thrombosis and Haemostasis ◽

10.1160/th12-02-0064 ◽

2012 ◽

Vol 108 (08) ◽

pp. 328-337 ◽

Cited By ~ 16

Author(s):

Maria Ersoy ◽

Paul Hjemdahl ◽

Naphtali Savion ◽

David Varon ◽

Galia Spectre ◽

...

Keyword(s):

Cell Adhesion ◽

B Cells ◽

Nk Cells ◽

Platelet Activation ◽

Platelet Adhesion ◽

Nk Cell ◽

Lymphocyte Adhesion ◽

Subendothelial Matrix ◽

Coated Surface ◽

Coated Surfaces

SummaryPlatelet adhesion at sites of cardiovascular injury may facilitate leukocyte deposition. We asked if and how platelets enhance lymphocyte adhesion on different subendothelial matrix protein (SEMP)-coated surface at arterial shear stress. Hirudinised whole blood was subjected to an arterial shear rate (500 s−1) in a Cone and Plate(let) analyser (CPA) for 5 minutes using plates coated with bovine serum albumin (BSA), collagen, fibrinogen, von Willebrand factor (vWF), or fibronectin. Platelet and lymphocyte adhesion were monitored by CPA and flow cytometry. Exposure of blood to collagen, fibrinogen, and vWF-coated surfaces induced platelet activation. The most marked effect was seen with collagen-coating, which markedly enhanced the adhesion of all lymphocyte subpopulations compared to BSA-coating. Fibrinogen-coating supported both T and NK cell adhesion, while vWF-coated surface only enhanced NK cell deposition. In contrast, fibronectin enhanced neither platelet activation nor lymphocyte adhesion. Moreover, platelets preferentially facilitated adhesion of large CD4+ and CD8+ T cells and NK cells, and of small B cells. Enhanced cell adhesion of larger lymphocytes was associated with elevated platelet conjugation and higher lymphocyte expression of PSGL-1, Mac-1, and CD40L. The enhancement of lymphocyte adhesion was totally platelet-dependent, and was abolished in platelet-depleted blood. Moreover, blockade of the platelet adhesion molecules P-selectin, GPIIb/IIIa, and CD40L attenuated platelet-dependent lymphocyte deposition. In conclusion, platelets support lymphocyte adhesion on SEMP-coated surfaces under arterial shear. The enhancement is selective for large T and NK cells and small B cells.

Download Full-text

The Role of LIM Kinase-1 in the Glycoprotein Ib-IX Complex-Mediated Platelet Activation and Thrombus Formation

Blood ◽

10.1182/blood.v112.11.112.112 ◽

2008 ◽

Vol 112 (11) ◽

pp. 112-112

Author(s):

Aleksandra Stojanovic ◽

Matvey Gorovoy ◽

Tatyana Voyno-Yasenetskaya ◽

Xiaoping Du

Keyword(s):

Shear Stress ◽

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Adhesion ◽

Wild Type ◽

Glycoprotein Ib ◽

Lim Kinase ◽

And Function

Abstract LIM Kinase (LIMK)-1 is a member of the LIMK family of serine-threonine protein kinases that phosphorylates actin-binding protein cofilin and regulates actin cytoskeleton organization. LIMK1 is expressed in many cell types including platelets but the exact role of LIMK1 in platelet function remains unclear. To determine the role of LIMK1 in platelet activation, wild type or LIMK1 knockout mouse platelets were stimulated with platelet agonists. Platelet aggregation and granule secretion were analyzed. Integrin-dependent second wave of platelet aggregation induced by von Willebrand factor (VWF) in the presence of VWF activator botrocetin was abolished in LIMK1 knockout platelets. In contrast, platelet aggregation in response to the agonist peptide of protease-activated receptor-4 (PAR4, thrombin receptor), ADP and collagen was either not affected or enhanced in LIMK1 knockout platelets in comparison with wild type mouse platelets. Thus, LIMK appears to play an important role in platelet activation stimulated by VWF binding to its platelet receptor, glycoprotein Ib-IX complex (GPIb-IX) but had no stimulatory effect on or negatively regulate the GPIb-IX-independent platelet activation pathways mediated by PAR-4, ADP receptors and collagen receptors. To determine whether ligand binding to GPIb-IX stimulates LIMK activation and function, platelets were stimulated with VWF in the presence of either ristocetin or botrocetin, and immunoblotted with antibodies specifically recognizing phosphorylated LIMK1 (Serine 505) or cofilin (Serine 3). VWF induced phosphorylation of LIMK1 and LIMK substrate cofilin. Thus, VWF indeed stimulates LIMK1 activation and function. An important physiological role of GPIb-IX in platelets is to mediate platelet adhesion to subendothelial-bound VWF under shear stress at sites of vascular injury. To determine whether LIMK1 is important in platelet adhesion, we investigated whether LIMK1 knockout affected platelet adhesion to VWF-coated surfaces. LIMK1 knockout platelets are defective in mediating stable platelet adhesion to vWF under shear stress, suggesting that LIMK1 plays an important role in GPIb signaling and GPIb-IX-mediated integrin activation that is required for stable platelet adhesion under shear stress. Importantly, LIMK1 knockout mice showed significant delay in the formation of occlusive thrombus following FeCl3-induced carotid artery injury in comparison with wild type mice, indicating that the role of LIMK1 in GPIb-IX-mediated platelet activation is important in in vivo thrombosis. Together, our study reveals that LIMK1 plays an important role in GPIb-IX-mediated platelet activation and arterial thrombosis in vitro and in vivo.

Download Full-text

Revised Model for Platelet Adhesion to Collagen.

Blood ◽

10.1182/blood.v114.22.2999.2999 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2999-2999

Author(s):

Lucia Stefanini ◽

Moritz Stolla ◽

Sean F Maloney ◽

Timothy Daniel Ouellette ◽

Claire Roden ◽

...

Keyword(s):

Platelet Activation ◽

Platelet Adhesion ◽

Thrombus Formation ◽

Mapk Signaling ◽

Low Flow ◽

Flow Conditions ◽

Integrin Activation ◽

Shear Rates ◽

Slow Kinetics ◽

Rap1 Activation

Abstract Abstract 2999 Poster Board II-968 The Gi-coupled ADP receptor, P2Y12, is the target of clopidogrel bisulfate (Plavix), currently the most successful anti-platelet strategy used in the clinic. In a recent study, we have shown that the Ca2+-sensing nucleotide exchange factor, CalDAG-GEFI, and P2Y12 represent the major signaling pathways leading to Rap1 and integrin activation in platelets (Cifuni et al., 2008, Blood). In the present study, we have further evaluated the importance of CalDAG-GEFI signaling and Rap1 activation for various aspects of platelet activation, and we have compared thrombus formation of CalDAG-GEFI−/− and WT/clopidogrel platelets under static and flow conditions in vitro. Our studies establish a revised model for platelet activation by collagen. In platelets activated with threshold concentrations of GPVI agonists, CalDAG-GEFI serves as a highly sensitive response element to Ca2+ that allows for the rapid activation of Rap1. CalDAG-GEFI-mediated Rap1 activation triggers a first wave of integrin activation and ERK (MAPK) signaling, followed by TxA2 release. TxA2 provides crucial feedback for the activation of PKC and granule/ADP release. ADP in turn triggers the second, P2Y12-dependent wave of Rap1-mediated signaling events, leading to the sustained activation of integrins and further release of TxA2. Higher concentrations of GPVI agonists lead to the concomitant activation of CalDAG-GEFI and PKC, facilitating platelet aggregation independent of feedback by endogenous TxA2. Under physiological flow conditions, CalDAG-GEFI-dependent platelet activation (clopidogrel-treated WT platelets) allowed for the formation of small but unstable thrombi, which rapidly disintegrated at high shear rates. In contrast, CalDAG-GEFI−/− platelets (P2Y12-dependent platelet activation) in anticoagulated blood firmly adhered to the thrombogenic surface but failed to form thrombi, even at high concentrations of collagen. Addition of exogenous TxA2 to anticoagulated CalDAG-GEFI−/− blood did not restore thrombus formation under flow. However, small thrombi were observed with non-anticoagulated CalDAG-GEFI−/− blood perfused at venous but not arterial shear rates, suggesting that a) locally generated thrombin facilitates the recruitment of free flowing CalDAG-GEFI−/− platelets to already adherent platelets, and b) the slow kinetics of P2Y12-dependent Rap1 activation only supports thrombin-induced platelet-platelet cohesion at low shear conditions. In conclusion, our studies demonstrate that CalDAG-GEFI/Rap1 signaling plays a critical role for the first wave of integrin activation and TxA2 generation important for platelet adhesion to a thrombogenic surface. Signaling by P2Y12/Rap1 is essential for sustained platelet activation/thrombus stabilization and partially compensates for CalDAG-GEFI/Rap1-mediated platelet adhesion under low flow conditions. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Anthocyanins Inhibit Platelet Activation and Attenuate Thrombus Growth In Both Human and Murine Thrombosis Models

Blood ◽

10.1182/blood.v116.21.3197.3197 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3197-3197 ◽

Cited By ~ 2

Author(s):

Yan Yang ◽

Zhenyin Shi ◽

Adili Reheman ◽

Wuxun Jin ◽

Conglei Li ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Function ◽

Intravital Microscopy ◽

Platelet Adhesion ◽

Thrombus Formation ◽

Vessel Occlusion ◽

Shear Rates ◽

Perfusion Chamber ◽

Platelet Adhesion And Aggregation

Abstract Abstract 3197 Background: Thrombosis and cardiovascular diseases (CVDs) result from blood vessel occlusion by inappropriately activated platelets. They are the leading causes of morbidity and mortality worldwide. Anthocyanins are major phytochemicals abundant in plant food and have been shown to play a protective role against CVDs. Our previous studies have demonstrated that anthocyanins are antioxidative and prevent inflammation (J Biol Chem. 2005; 280:36792-01; Arterioscler Thromb Vasc Biol. 2007; 27:519-24), which may indirectly affect platelet function. It has also been reported that anthocyanins affect platelet activities in whole blood and platelet rich plasma (PRP). However, the direct effects of anthocyanins on platelet function and thrombus formation have not been studied. Methods: Here we investigated the effects of anthocyanins on thrombosis using purified platelets as well as several thrombosis models in vitro and in vivo. Cyaniding-3-gulucoside (Cy-3-g) and delphinidin-3-glucoside (Dp-3-g), the two predominantly bioactive compounds of anthocyanin preparations, were prepared from Polyphenol AS Company in Norway. Purified gel-filtered platelets and PRP from healthy human volunteers and C57BL/6J mice were incubated at 37°C for 10 minutes with different concentrations (0.5μM, 5μM and 50μM) of Cy-3-g, Dp-3-g or PBS buffer as a control. Platelet aggregation was assessed by aggregometry using 5μM ADP, 10μg/ml collagen, or 100μM thrombin receptor activating peptide (TRAP; AYPGKF) as agonists. Platelet adhesion and aggregation were assessed in response to an immobilized collagen matrix in an ex vivo perfusion chamber at both high (1800 s-1) and low (600 s-1) shear rates. The expression of activated GPIIbIIIa was determined via PAC-1 monoclonal antibody in flow cytometry. Lastly, the effects of anthocyanins on thrombus formation in C57BL/6J mice were assessed using a FeCl3-induced intravital microscopy thrombosis model. Results: Both Cy-3-g and Dp-3-g significantly inhibited platelet aggregation induced by collagen and TRAP in gel-filtered platelets, and inhibited aggregation induced by ADP, TRAP and collagen in human and mouse PRP. These inhibitory functions were observed at Cy-3-g and Dp-3-g doses as low as 0.5μM. Cy-3-g and Dp-3-g also reduced the surface expression of activated GPIIbIIIa on resting human platelets in a dose-dependent manner. These compounds also markedly reduced platelet adhesion and aggregation in perfusion chamber assays at both low and high shear rates. Using intravital microscopy, we further demonstrated that Cy-3-g and Dp-3-g decreased platelet deposition, destabilized thrombi, and prolonged the time required for thrombus formation and vessel occlusion. Conclusions: our data clearly demonstrated for the first time that anthocyanin compounds directly inhibited platelet activation, adhesion and aggregation, as well as attenuated thrombus growth at both arterial and veinous shear stresses. These effects on platelets likely contribute to the protective effects of anthocyanins against thrombosis and CVDs. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Potentiation by adrenaline of human platelet activation and the inhibition by the alpha-adrenergic antagonist nicergoline of platelet adhesion, secretion and aggregation

Agents and Actions ◽

10.1007/bf01964968 ◽

1986 ◽

Vol 18 (5-6) ◽

pp. 586-595 ◽

Cited By ~ 14

Author(s):

François Lanza ◽

Jean-Pierre Cazenave ◽

Alain Beretz ◽

Astrid Sutter-Bay ◽

Jean-Georges Kretz ◽

...

Keyword(s):

Platelet Activation ◽

Platelet Adhesion ◽

Human Platelet ◽

Adrenergic Antagonist

Download Full-text

Effects of ε-Aminocaproic Acid and Aprotinin on Leukocyte–Platelet Adhesion in Patients Undergoing Cardiac Surgery

Anesthesiology ◽

10.1097/00000542-200402000-00008 ◽

2004 ◽

Vol 100 (2) ◽

pp. 225-233 ◽

Cited By ~ 22

Author(s):

Philip E. Greilich ◽

Chad F. Brouse ◽

Christine S. Rinder ◽

Brian R. Smith ◽

Bernardo A. Sandoval ◽

...

Keyword(s):

Cardiac Surgery ◽

Platelet Activation ◽

Platelet Adhesion ◽

Relative Effectiveness ◽

Aminocaproic Acid ◽

Saline Control ◽

Plasmin Activity ◽

Double Blind ◽

Cd11b Expression ◽

D Dimer

Background The administration of aprotinin during cardiopulmonary bypass (CPB) is hypothesized to decrease activation of leukocytes and platelets and possibly reduce their adhesion. Although epsilon-aminocaproic acid (EACA) shares the ability of aprotinin to inhibit excessive plasmin activity after CPB, its effect on leukocyte and platelet activation and leukocyte-platelet (heterotypic) adhesion is largely unknown. This study was performed to determine the relative effectiveness of the antifibrinolytics, aprotinin and EACA, at reducing leukocyte and platelet activation and leukocyte-platelet conjugate formation in patients undergoing CPB. Methods Thirty-six patients scheduled to undergo cardiac surgery with CPB were randomized in a double-blind fashion to receive EACA, aprotinin, or saline (placebo). Markers of plasmin activity (D-dimer concentrations), platelet activation (CD62P), leukocyte activation (CD11b), and leukocyte-platelet adhesion (monocyte- and neutrophil-platelet conjugates) were measured before, during, and after CPB. Results Platelet CD62P (P-selectin), monocyte CD11b, and monocyte-platelet conjugates were all significantly increased (compared with baseline) in the saline group during and after CPB. Despite equivalent reductions in D-dimer formation in patients receiving EACA (P < 0.0001) and aprotinin (P < 0.0001), decreases in platelet CD62P and monocyte CD11b expression were incomplete (not significantly different from saline control). In contrast, peak monocyte-platelet conjugate formation was significantly reduced by both EACA (P = 0.026) and aprotinin (P = 0.039) immediately after CPB. Conclusions EACA seems to be as effective as aprotinin at reducing peak monocyte-platelet adhesion after CPB. Furthermore, inhibition of excessive plasmin activity seems to influence monocyte-platelet adhesion. The findings suggest that monocyte-platelet conjugate formation may be a useful marker of monocyte and platelet activation in this clinical setting.

Download Full-text

Distinct determinants on collagen support alpha 2 beta 1 integrin-mediated platelet adhesion and platelet activation.

Cell Regulation ◽

10.1091/mbc.2.11.905 ◽

1991 ◽

Vol 2 (11) ◽

pp. 905-913 ◽

Cited By ~ 78

Author(s):

S A Santoro ◽

J J Walsh ◽

W D Staatz ◽

K J Baranski

Keyword(s):

Platelet Activation ◽

Platelet Adhesion ◽

Side Chains ◽

Amino Groups ◽

Chemical Modifications ◽

Platelet Surface ◽

Site Model ◽

Amino Acid Side Chains ◽

Beta 1 Integrin ◽

Collagen Receptor

Recent studies have revealed that the sequence of amino acids asp-gly-glu-ala represents an essential determinant of the site within the alpha 1(I)-CB3 fragment of collagen recognized by the alpha 2 beta 1 integrin cell surface collagen receptor (Staatz et al., 1991). Studies employing chemical modifications of collagen amino acid side chains confirm both the essential nature of the acidic side chains of aspartic acid and glutamic acid residues and the nonessentiality of lysine epsilon-amino groups in supporting adhesion mediated by the alpha 2 beta 1 integrin. The approach also indicates the presence of a distinct determinant on collagen separate from the alpha 2 beta 1 recognition site that contains essential lysine side chains and that is necessary for subsequent interactions with the platelet surface that give rise to collagen-induced platelet activation and secretion. The two-step, two-site model for cellular signaling involving both an integrin and a signal-transducing coreceptor suggested by these data may be common to other integrin-mediated processes.

Download Full-text