Microfluidic focal thrombosis model for measuring murine platelet deposition and stability: PAR4 signaling enhances shear‐resistance of platelet aggregates

Although much is known about extrinsic regulators of platelet function such as nitric oxide and prostaglandin I 2 (PGI 2 ), considerably less is known about intrinsic mechanisms that prevent overly robust platelet activation after vascular injury. Here we provide the first evidence that regulators of G-protein signaling (RGS) proteins serve this role in platelets, using mice with a G184S substitution in G i2␣ that blocks RGS/G i2 interactions to examine the consequences of lifting constraints on G i2 -dependent signaling with- IntroductionThe hemostatic response to injury in humans and other species with a closed, high-pressure circulatory system represents a balance between minimizing blood loss and avoiding vascular occlusion. How this balance is achieved is only partially understood. Hemostatic thrombi are formed by a combination of fibrin and platelets. While fibrin deposition is controlled by regulating production of thrombin and subsequently by protease inhibitors, platelet activation is controlled by limiting the availability of platelet agonists and by releasing inhibitors such as prostaglandin I 2 (PGI 2 ) and nitric oxide from endothelial cells. PGI 2 and nitric oxide prevent unwarranted platelet activation by raising basal cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) levels. Coming from a source outside of the platelet, they can be viewed as extrinsic regulators of platelet function. Here we asked whether platelet activation is also subject to autonomous (ie, intrinsic) regulation that limits the extent of platelet accumulation by limiting internal platelet signaling.Although there are multiple ways that intrinsic regulation could be accomplished, most platelet agonists activate members of the G protein-coupled receptor family, making G proteins a logic target for regulation. In general, G proteins remain active until hydrolysis of G ␣ -bound guanosine-5Ј-triphosphate (GTP) restores the resting state. The rate of inactivation is determined in part by members of the regulators of G-protein signaling (RGS) family, which accelerate GTP hydrolysis by G ␣ . [1][2][3][4] The 37 known RGS proteins have a 120 amino acid RGS domain that can interact with the switch region of activated G ␣ . 5,6 As many as 10 RGS proteins have been reported in platelets, although many of them solely at the level of RNA transcripts. [7][8][9][10][11][12] Nothing is known about the impact of these proteins on platelet activation in vitro or in vivo.Here we asked whether RGS proteins limit platelet accumulation during thrombus formation and, if so, whether this is through an effect on the initiation or the magnitude of the platelet response to agonists. Because of uncertainty about the full repertoire of RGS proteins expressed in platelets, the paucity of available RGS protein gene knockouts, and ambiguities about the specificity of RGS/G ␣ interactions, we studied mice in which a substitution (G184S) in the ␣ subunit of G i2 renders it resistant to accelerated turn-off by all RGS proteins. 13 T...

Section: Flow Chamber Studiesmentioning

confidence: 99%

RGS/Gi2α interactions modulate platelet accumulation and thrombus formation at sites of vascular injury

Signarvic¹,

Cierniewska²,

Stalker³

et al. 2010

“…A volume of 5 mL of collagen was perfused through the patterning channel (250 mm wide 3 60 mm high) of a microfluidic device to create a single stripe of fibrillar collagen, as previously described. 38 Lipidated TF was then sorbed to the collagen surface by introduction of 5 mL of Dade Innovin PT reagent (20 nM stock concentration) 39 diluted 300-, 100-, and fivefold with N-2-hydroxyethylpiperazine-N9-2-ethanesulfonic acid buffered saline to obtain low, medium, and high TF surface concentrations ([TF] wall ) of ;0.1, ;0.2, and ;2 molecules per mm 2 , respectively, as estimated by imaging of sorbed annexin V-fluorescein isothiocyanate-stained vesicles (supplemental Figure 1). In all experiments, the Dade Innovin PT reagent was incubated with the collagen for 30 minutes without flow and then rinsed and blocked with 20 mL 0.1% bovine serum albumin buffer.…”

Section: Methodsmentioning

confidence: 99%

FXIa and platelet polyphosphate as therapeutic targets during human blood clotting on collagen/tissue factor surfaces under flow

Zhu

Travers

Morrissey

et al. 2015

“…Fabrication of microfluidic devices and microfluidic collagen patterning were performed as previously described. 24 Briefly, a 100-m strip of fibrillar collagen type I (200 g/mL) was deposited and immobilized by microfluidic patterning along the length of a glass slide. A poly dimethylsiloxane device with 10 flow channels (width: 250 m, height: 60 m, length: 6 mm) was oriented perpendicularly to the patterned collagen.…”

Section: Gfp Expression In Blood Plateletsmentioning

confidence: 99%

The kinetics of αIIbβ3 activation determines the size and stability of thrombi in mice: implications for antiplatelet therapy

Stolla

Stefanini

Roden

et al. 2011

115

Two major pathways contribute to Rasproximate-1-mediated integrin activation in stimulated platelets. Calcium and diacyglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI, Ras-GRP2) mediates the rapid but reversible activation of integrin ␣IIb␤3, while the adenosine diphosphate receptor P2Y12, the target for antiplatelet drugs like clopidogrel, facilitates delayed but sustained integrin activation. To establish CalDAG-GEFI as a target for antiplatelet therapy, we compared how each pathway contributes to thrombosis and hemostasis in mice. Ex vivo, thrombus formation at arterial or venous shear rates was markedly reduced in CalDAG-GEFI ؊/؊ blood, even in the presence of exogenous adenosine diphosphate and thromboxane A 2 . In vivo, thrombosis was virtually abolished in arterioles and arteries of CalDAG-GEFI ؊/؊ mice, while small, hemostatically active thrombi formed in venules. Specific deletion of the C1-like domain of CalDAG-GEFI in circulating platelets also led to protection from thrombus formation at arterial flow conditions, while it only marginally increased blood loss in mice. In comparison, thrombi in the micro-and macrovasculature of clopidogrel-treated wild-type mice grew rapidly and frequently embolized but were hemostatically inactive. Together, these data suggest that inhibition of the catalytic or the C1 regulatory domain in CalDAG-GEFI will provide strong protection from atherothrombotic complications while maintaining a better safety profile than P2Y12 inhibitors like clopidogrel. (Blood. 2011; 117(3):1005-1013) IntroductionArterial thrombosis in the coronary or cerebrovascular circulation is the principal pathological process underlying acute coronary syndrome and ischemic stroke, which together represent the leading cause of morbidity and mortality in industrialized countries. 1 Platelet activation is a central event in the pathogenesis of arterial thrombosis. Currently, the most powerful antiplatelet agents used in the clinic are inhibitors of cyclooxygenase-1 (acetylsalicylic acid, aspirin), the platelet adenosine diphosphate (ADP) receptor P2Y12 (eg, clopiodgrel or Plavix), and integrin ␣IIb␤3 (eg, abciximab or Reopro). 2,3 These agents have all been shown to improve clinical outcomes in large-scale randomized controlled trials. However, all therapies have limitations that include uncertainty about optimal dosing, questions about resistance, and issues regarding the lack of reversibility in situations where bleeding risks are high.␣IIb␤3, the platelet fibrinogen receptor, is the best-studied member of the integrin family. 4,5 Like most integrins, especially those regulating adhesion and trafficking of blood cells, it is expressed in a low-affinity state on resting platelets. Engagement of agonist receptors on the platelet surface triggers intracellular signaling events, which lead to inside-out activation of ␣IIb␤3. Deficiency in ␣IIb␤3 completely inhibits the ability of platelets to aggregate and adhere to sites of injury. 6,7 Consequently, inhibitors to integrin ␣IIb␤3 show...