MyosinIIa contractility is required for maintenance of platelet structure during spreading on collagen and contributes to thrombus stability

Calaminus, Simon D. J.; Auger, Jocelyn M.; McCarty, OJ; Wakelam, Michael J.O.; Machesky, Laura M.; Watson, Steve P.

doi:10.1111/j.1538-7836.2007.02696.x

Cited by 63 publications

(72 citation statements)

References 35 publications

(62 reference statements)

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“…The observation that loss of aggregate stability in the presence of either Src kinase blockade or actin disruption is associated with retraction of lamellipodia and the appearance of single rounded platelets leads us to speculate that Src kinases contribute to aggregate stability through the actin cytoskeleton, especially bearing in mind the wealth of evidence for direct regulation of actin polymerization by integrin ␣IIb␤3 and GPVI. 8,16,17 Several group have reported stable thrombus formation at arteriolar rates of shear in vitro, as observed in the present study. 15,18,19 On the other hand, over the past few years, the use of various in vivo thrombosis models has revealed unstable aggregate formation in the absence of many platelet receptors or receptor mutants, including the ␣ 2A -adrenergic receptor, SLAM receptors, CD40L, and the diYF mutant of the integrin ␤3 subunit.…”

Section: Discussionsupporting

confidence: 77%

“…7 Further, inhibition of stress fiber formation using selective inhibitors or myosin IIa-deficient mice leads to reduced aggregate formation on collagen at arteriolar shear. 8,9 Rac and myosin IIa also contribute to thrombus stability in vivo, although this may additionally reflect their role in clot retraction. 8 -10 Collagen signaling is maintained over several hours in platelets and is required for sustained lamellipodia formation in vitro.…”

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confidence: 99%

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Dynamic Tyrosine Kinase-Regulated Signaling and Actin Polymerisation Mediate Aggregate Stability Under Shear

Auger

Watson

2008

ATVB

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Objective-Aggregate formation on collagen at arteriolar rates of shear is mediated by coordinated signaling between tyrosine kinase-linked and G protein-coupled receptors. We have investigated the role of these receptors and the actin cytoskeleton in maintaining aggregate stability under shear. Methods and Results-Platelet aggregates are rapidly formed when blood is flowed over collagen at 1000 s Ϫ1 and remain stable over 20 minutes. A novel fibrin-independent mechanism of retraction against the direction of flow occurs at the aggregate front and recruits platelets into the main aggregate. Stable aggregates are not observed in the presence of cytochalasin D, which blocks de novo actin polymerization. When exposed to the Src family kinase inhibitor, PD0173952, preformed aggregates spread in the direction of flow and rounded platelets appear within the aggregate body and are lost in the direction of flow. A similar set of observations is observed in the presence of latrunculin A, which disrupts preexisting actin filaments, but not in the combined presence of inhibitors of ADP and thromboxane A 2 formation. Conclusions-Maintenance of stable aggregates at high shear is a dynamic process mediated by Src kinases and actin polymerization. These signals maintain aggregates in a compact structure and prevent continuous streaming of platelets. Key Words: platelet aggregation Ⅲ collagen Ⅲ cytoskeleton Ⅲ Src kinase Ⅲ shear C ollagen is a key component of the vascular basement membrane that surrounds blood vessels and the deeper vessel wall. On injury, the interaction of platelets with collagen plays a critical role in initiating thrombus formation and preventing excessive blood loss. Collagen is also a major component of atherosclerotic plaques and mediates the massive platelet activation that occurs on plaque rupture, leading to thrombotic disorders such as myocardial infarction and stroke.The events underlying aggregate formation on collagen at arteriolar rates of shear in vitro have been studied extensively. Platelet tethering or capture is mediated by binding of collagen-adherent von Willebrand factor (vWF) to the glycoprotein (GP) Ib-IX-V complex. Subsequent activation via the collagen receptor GPVI converts integrins ␣IIb␤3 and ␣2␤1 to high-affinity states that support binding to vWF and collagen, respectively, giving rise to stable adhesion. 1,2 Under these conditions, GPVI is critical for aggregate formation, 3,4 whereas the role of ␣2␤1 is supportive because of redundancy with ␣IIb␤3. 5 The secondary mediators, ADP and thromboxane A 2 , support aggregate growth through activation of integrin ␣IIb␤3 and subsequent crosslinking via vWF and fibrinogen. The aggregate is further stabilized by "late-stage signaling events" through a variety of surface proteins, including integrins, ephrins and Eph kinases, cell adhesion molecules (eg, ESAM and JAM-A), and semaphorin 4D. 6 The presence of anticoagulant prevents conversion of fibrinogen to fibrin and further stabilization through clot retraction.The actin cytoskeleton has ...

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Section: Discussionsupporting

confidence: 77%

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confidence: 99%

Dynamic Tyrosine Kinase-Regulated Signaling and Actin Polymerisation Mediate Aggregate Stability Under Shear

Auger

Watson

2008

ATVB

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“…Considering these differences in traction forces, we conclude that the mechanism to generate traction forces may depend on the cell type, possibly involving fluid flows and cytoskeletal disassembly processes apart from the actomyosin mechanism. Interestingly, it has been found that myosin contractility is required for maintenance of platelet structure during spreading (Calaminus et al, 2007). Future experiments, investigating the role of actomyosin in platelets, could be performed by adding blebbistatin or the rho kinase inhibitor Y27632.…”

Section: Discussionmentioning

confidence: 99%

Force field evolution during human blood platelet activation

Henriques

Sandmann

Strate

et al. 2012

Journal of Cell Science

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SummaryContraction at the cellular level is vital for living organisms. The most prominent type of contractile cells are heart muscle cells, a lesswell-known example is blood platelets. Blood platelets activate and interlink at injured blood vessel sites, finally contracting to form a compact blood clot. They are ideal model cells to study the mechanisms of cellular contraction, as they are simple, having no nucleus, and their activation can be triggered and synchronized by the addition of thrombin. We have studied contraction using human blood platelets, employing traction force microscopy, a single-cell technique that enables time-resolved measurements of cellular forces on soft substrates with elasticities in the physiological range (,4 kPa). We found that platelet contraction reaches a steady state after 25 min with total forces of ,34 nN. These forces are considerably larger than what was previously reported for platelets in aggregates, demonstrating the importance of a single-cell approach for studies of platelet contraction. Compared with other contractile cells, we find that platelets are unique, because force fields are nearly isotropic, with forces pointing toward the center of the cell area.

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“…[19][20][21] Nonetheless, Rho kinase appears to play an important role in platelet function, by promoting focal adhesion-like complexes 18 and actin stress fibers in spreading platelets. 22 Such structures may play a key role in regulating the adhesive function of platelets, as inhibition of Rho kinase undermines the stability of platelet-matrix and plateletplatelet interactions in a shear field, 23 leading to a major defect in thrombus growth. 22 Genetic abnormalities in myosin IIA (termed the MYH9 disorders) encompass several autosomal dominant disorders, including the May-Hegglin anomaly and Epstein, Sebastian, and Fechtner syndromes.…”

Section: Introductionmentioning

confidence: 99%

“…22 Such structures may play a key role in regulating the adhesive function of platelets, as inhibition of Rho kinase undermines the stability of platelet-matrix and plateletplatelet interactions in a shear field, 23 leading to a major defect in thrombus growth. 22 Genetic abnormalities in myosin IIA (termed the MYH9 disorders) encompass several autosomal dominant disorders, including the May-Hegglin anomaly and Epstein, Sebastian, and Fechtner syndromes. 24,25 A hallmark feature of these disorders is the failure of platelets to undergo shape change following soluble agonist stimulation, whereas platelet aggregation remains intact.…”

Section: Introductionmentioning

confidence: 99%

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

Ono

Westein

Hsiao

et al. 2008

Blood

126

106

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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. Realtime 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. IntroductionThe ability of platelets to adhere to subendothelial matrix proteins and with other platelets at sites of vascular injury is critical for hemostatic plug formation and for the development of arterial thrombi. 1,2 The hemostatic response can be divided into 2 temporally distinct phases: the primary hemostatic plug, composed of aggregated platelets that form independent of fibrin formation 1 ; and the secondary hemostatic response, wherein fibrin polymers enmeshed into the developing thrombus physically stabilize the platelet plug. 3 During hemostatic plug formation, platelets undergo a complex series of morphologic and functional responses that require extensive remodeling of the actin cytoskeleton. These cytoskeletal changes are indispensable for the normal hemostatic function of platelets and are controlled by complex network of signaling, structural, and regulatory proteins. 4 The actin-based cytoskeleton can be separated into 2 functionally distinct structures: (1) the spectrin-rich membrane skeleton, lining the inner plasma membrane, and (2) the cytoskeleton, consisting of long actin filaments that radiate from the cell center to the surface membrane. 4,5 The membrane skeleton is essential for maintaining the structure and integrity of the surface membrane, whereas the cytoskeleton, through its attachment to myosin, principally generates contractile forces within the cell. 6 The internal generation of contractile force has a well-defined role in regulating platelet shape change 7,8 and in promoting granule secretion, 9,10 whereas the extracellular transmission of cytoskeletal contractile force i...

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MyosinIIa contractility is required for maintenance of platelet structure during spreading on collagen and contributes to thrombus stability

Cited by 63 publications

References 35 publications

Dynamic Tyrosine Kinase-Regulated Signaling and Actin Polymerisation Mediate Aggregate Stability Under Shear

Dynamic Tyrosine Kinase-Regulated Signaling and Actin Polymerisation Mediate Aggregate Stability Under Shear

Force field evolution during human blood platelet activation

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

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