From www.bloodjournal.org by guest on September 11, 2016. For personal use only.
11 SEPTEMBER 2014
I VOLUME 124, NUMBER 11
l l l THROMBOSIS & HEMOSTASIS
Comment on Ivanciu et al, page 1705; Welsh et al, page 1808; Tomaiuolo et al, page 1816; and Stalker et al, page 1824
Thrombus formation reimagined ----------------------------------------------------------------------------------------------------Robert Flaumenhaft
BETH ISRAEL DEACONESS MEDICAL CENTER; HARVARD MEDICAL SCHOOL
In this issue of Blood, Ivanciu et al, Welsh et al, Tomaiuolo et al, and Stalker et al, in a series of studies from the University of Pennsylvania, use intravital microscopy to evaluate how thrombi form following vascular injury and reveal unexpected interactions and physical forces that dictate clot initiation and architecture.1-4
I
n 1882, Dr Giulio Bizzozero described the use of intravital microscopy to detect the accumulation of platelets following needleinduced injury of the mesenteric artery in
rabbits and guinea pigs.5 Bizzozero’s paper was not only the first to define the platelet in name (“Blutpl¨attchen” in German) but was also the first to functionally define platelets as the
Thrombus formation following penetrating injury. In this depiction of thrombus formation induced by laser or sharpened glass probe, injury results in the exposure of phosphatidylserine on the endothelial cell surface and initiates assembly of the prothrombinase complex. A core of tightly packed platelets (blue) forms at the injury site and restricts the transport of agonists, including thrombin. A shell of loosely adherent, unactivated platelets (red) forms around the core. The core expands over time as agonists diffuse out of the core into the shell, creating a transition zone (dotted lines) in which activation of loosely adherent platelets occurs. Professional illustration by Luk Cox, Somersault1824.
BLOOD, 11 SEPTEMBER 2014 x VOLUME 124, NUMBER 11
primary cell type contributing to thrombus formation. Over the past decade, the study of thrombus formation using intravital microscopy has been transformed by the use of high-speed, near-simultaneous acquisition to image multiple fluorescent probes during thrombus formation in vivo.6,7 Real-time imaging of platelets, leukocytes, endothelial cells, tissue factor, fibrin, phosphatidylserine, and plasma proteins in living mice has revolutionized our understanding of how vascular injury rapidly evokes clot formation. These in vivo approaches have served as an important counterpoint to in vitro studies performed on isolated platelets, sometimes confirming in vitro findings, but often not. Studies from the University of Pennsylvania laboratories of Drs Rodney Camire and Skip Brass now provide exceptional examples of how intravital experiments can contradict longstanding dogma derived from ex vivo evaluation of isolated platelets and how a systems approach assessing the clot as a functional unit can reveal information not attainable by studying isolated platelets. In the first article, Ivanciu et al debunk the long-held view that activated platelets provide the primary surface for assembly of the prothrombinase complex.1 They infused site-specifically labeled factors Va and Xa into hemophilia B mice to evaluate the localization of the prothrombinase complex during thrombus formation. The observation that factor Va corrects the thrombotic defect in the hemophilia B mice proves that it localizes to areas of clotting in this model.8 The authors found that the prothrombinase complex assembled largely on damaged endothelium and not on activated platelets (see figure). Preventing platelet accumulation at the injury site had little effect on prothrombinase formation. Consistent with previous reports, the investigators showed that inhibition of platelet accumulation did not affect fibrin generation following vascular injury.9 To verify endothelial localization, the authors
1697
From www.bloodjournal.org by guest on September 11, 2016. For personal use only.
infused labeled factor Xa into mice that express green fluorescent protein (GFP) under the endothelial cell-specific Tie2 promoter. These studies confirmed that factor Xa associates with endothelial cell surfaces. Further studies showed that factor Xa localizes to areas of phosphatidylserine exposure on injured endothelium. This work defines a pathway whereby injury-dependent phosphatidylserine exposure on endothelium leads to FVa and FXa binding with subsequent fibrin generation and shows that, although platelets are able to support fibrin formation in vitro, the endothelium provides the surface for assembly of the prothrombinase complex in this in vivo model. Beyond showing how thrombi are initiated, new studies from investigators at the University of Pennsylvania reveal how platelet-rich thrombi grow and achieve their characteristic architecture. Previous work from this group demonstrated that platelet-rich thrombi consists of 2 zones: a core of tightly packed, activated platelets and a shell of loosely packed, unactivated platelets.10 In their new study, Welsh et al showed that the transport of diffusible agonists was slower in the core of the thrombus and faster in the shell.2 As an example, they showed that thrombin diffuses very little from the thrombus core, which is juxtaposed to the damaged endothelium. This arrangement sets up a transition zone in which thrombin (and other agonists) at the edge of the core activates resting platelets in the adjacent edge of the shell region (see figure). In this manner, the core grows over time. These studies demonstrate that the degree of platelet activation required to elicit granule release does not occur at the periphery of the outer shell, which is exposed to flowing blood, but rather at its core. An important correlate of this observation is that platelets accumulating in thrombi release their contents not into flowing blood but rather into the body of the thrombus. What are the consequences of releasing soluble agonists into the thrombus rather than into flowing blood? Tomaiuolo et al use a computational approach to show that the effect is profound.3 They modeled the movement of soluble agonists in a thrombus and showed that convection had very little influence on the movement of agonists released
1698
into a thrombus. Instead, the movement of these agonists was controlled by diffusion within the body of the thrombus. Modeling suggested that intrathrombus plasma velocity was ;1000-fold slower than bulk velocity in the vessel and that this “solute haven” is achieved in surprisingly small thrombi (16 platelets). The rate of diffusion is critically regulated by the packing density of platelets, which can be measured by the size of the gaps between platelets. If packing density is an important determinant of agonist diffusion in a thrombus, then modulation of packing density should have substantial effects on thrombus growth and architecture. This prediction is supported by computation studies and is validated in a mouse model in which packing density is impaired. Stalker et al studied thrombus formation and solute transport in the diYF mouse4 in which tyrosines in the b tail of aIIbb3 are mutated to phenylalanine, resulting in a defect in outside-in signaling.11 Although platelets from these mice exhibited normal fibrinogen binding and granule release, they had a defect in clot retraction that led to lower packing densities. This defect increased intrathrombus transport rates and decreased thrombin and platelet activity within the thrombus core because thrombin leached out of thrombi more quickly in diYF mice compared with wild-type controls. These results confirm that gap size, which is increased in diYF mice, is a critical determinant of solute transport and, in turn, agonist distribution and platelet activation within the thrombus. This series of articles raises important questions for the evaluation of existing antiplatelet agents and the design of novel antithrombotics. Might indications for shelltargeted antiplatelet agents (eg, aspirin, clopidogrel) differ from those for core-targeted agents (eg, hirudin, vorapaxar)? Might reagents targeting platelet packing density rather than platelet activation demonstrate better risk-benefit ratios in clinical use? In commenting on previous attempts to define blood platelets in his famous 1882 publication, Giulio Bizzozero noted that, “It is astonishing that none of the previous
investigators made use of the observation of circulating blood in living animals.”5 One hundred thirty years later, the strategy of looking closely at platelets in vivo and considering how they act as a unit, rather studying them in isolation, continues to provide information beyond that obtained by the evaluation of individual platelets. This approach will serve to improve our understanding of how thrombi form, expand, and ultimately resolve. Conflict-of-interest disclosure: The author declares no competing financial interests. n REFERENCES 1. Ivanciu L, Krishnaswamy S, Camire RM. New insights into the spatiotemporal localization of prothrombinase in vivo. Blood. 2014;124(11):1705-1714. 2. Welsh JD, Stalker TJ, Voronov R, et al. A systems approach to hemostasis: 1. The interdependence of thrombus architecture and agonist movements in the gaps between platelets. Blood. 2014;124(11):1808-1815. 3. Tomaiuolo M, Stalker TJ, Welsh JD, Diamond SL, Sinno T, Brass LF. A systems approach to hemostasis: 2. Computational analysis of molecular transport in the thrombus microenvironment. Blood. 2014;124(11): 1816-1823. 4. Stalker TJ, Welsh JD, Tomaiuolo M, et al. A systems approach to hemostasis: 3. Thrombus consolidation regulates intrathrombus solute transport and local thrombin activity. Blood. 2014;124(11):1824-1831. 5. Ribatti D, Crivellato E. Giulio Bizzozero and the discovery of platelets. Leuk Res. 2007;31(10):1339-1341. 6. Falati S, Gross P, Merrill-Skoloff G, Furie BC, Furie B. Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse. Nat Med. 2002;8(10):1175-1181. 7. Celi A, Merrill-Skoloff G, Gross P, et al. Thrombus formation: direct real-time observation and digital analysis of thrombus assembly in a living mouse by confocal and widefield intravital microscopy. J Thromb Haemost. 2003; 1(1):60-68. 8. Schlachterman A, Schuettrumpf J, Liu J-H, et al. Factor V Leiden improves in vivo hemostasis in murine hemophilia models. [published correction appears in J Thromb Haemost. 2006;4(4):925]. J Thromb Haemost. 2005; 3(12):2730-2737. 9. Vandendries ER, Hamilton JR, Coughlin SR, Furie B, Furie BC. Par4 is required for platelet thrombus propagation but not fibrin generation in a mouse model of thrombosis. Proc Natl Acad Sci USA. 2007;104(1):288-292. 10. Stalker TJ, Traxler EA, Wu J, et al. Hierarchical organization in the hemostatic response and its relationship to the platelet-signaling network. Blood. 2013;121(10): 1875-1885. 11. Law DA, DeGuzman FR, Heiser P, Ministri-Madrid K, Killeen N, Phillips DR. Integrin cytoplasmic tyrosine motif is required for outside-in alphaIIbbeta3 signalling and platelet function. Nature. 1999;401(6755):808-811. © 2014 by The American Society of Hematology
BLOOD, 11 SEPTEMBER 2014 x VOLUME 124, NUMBER 11
From www.bloodjournal.org by guest on September 11, 2016. For personal use only.
2014 124: 1697-1698 doi:10.1182/blood-2014-06-579656
Thrombus formation reimagined Robert Flaumenhaft
Updated information and services can be found at: http://www.bloodjournal.org/content/124/11/1697.full.html Articles on similar topics can be found in the following Blood collections Free Research Articles (4037 articles) Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.
11 SEPTEMBER 2014
I VOLUME 124, NUMBER 11
l l l THROMBOSIS & HEMOSTASIS
Comment on Ivanciu et al, page 1705; Welsh et al, page 1808; Tomaiuolo et al, page 1816; and Stalker et al, page 1824
Thrombus formation reimagined ----------------------------------------------------------------------------------------------------Robert Flaumenhaft
BETH ISRAEL DEACONESS MEDICAL CENTER; HARVARD MEDICAL SCHOOL
In this issue of Blood, Ivanciu et al, Welsh et al, Tomaiuolo et al, and Stalker et al, in a series of studies from the University of Pennsylvania, use intravital microscopy to evaluate how thrombi form following vascular injury and reveal unexpected interactions and physical forces that dictate clot initiation and architecture.1-4
I
n 1882, Dr Giulio Bizzozero described the use of intravital microscopy to detect the accumulation of platelets following needleinduced injury of the mesenteric artery in
rabbits and guinea pigs.5 Bizzozero’s paper was not only the first to define the platelet in name (“Blutpl¨attchen” in German) but was also the first to functionally define platelets as the
Thrombus formation following penetrating injury. In this depiction of thrombus formation induced by laser or sharpened glass probe, injury results in the exposure of phosphatidylserine on the endothelial cell surface and initiates assembly of the prothrombinase complex. A core of tightly packed platelets (blue) forms at the injury site and restricts the transport of agonists, including thrombin. A shell of loosely adherent, unactivated platelets (red) forms around the core. The core expands over time as agonists diffuse out of the core into the shell, creating a transition zone (dotted lines) in which activation of loosely adherent platelets occurs. Professional illustration by Luk Cox, Somersault1824.
BLOOD, 11 SEPTEMBER 2014 x VOLUME 124, NUMBER 11
primary cell type contributing to thrombus formation. Over the past decade, the study of thrombus formation using intravital microscopy has been transformed by the use of high-speed, near-simultaneous acquisition to image multiple fluorescent probes during thrombus formation in vivo.6,7 Real-time imaging of platelets, leukocytes, endothelial cells, tissue factor, fibrin, phosphatidylserine, and plasma proteins in living mice has revolutionized our understanding of how vascular injury rapidly evokes clot formation. These in vivo approaches have served as an important counterpoint to in vitro studies performed on isolated platelets, sometimes confirming in vitro findings, but often not. Studies from the University of Pennsylvania laboratories of Drs Rodney Camire and Skip Brass now provide exceptional examples of how intravital experiments can contradict longstanding dogma derived from ex vivo evaluation of isolated platelets and how a systems approach assessing the clot as a functional unit can reveal information not attainable by studying isolated platelets. In the first article, Ivanciu et al debunk the long-held view that activated platelets provide the primary surface for assembly of the prothrombinase complex.1 They infused site-specifically labeled factors Va and Xa into hemophilia B mice to evaluate the localization of the prothrombinase complex during thrombus formation. The observation that factor Va corrects the thrombotic defect in the hemophilia B mice proves that it localizes to areas of clotting in this model.8 The authors found that the prothrombinase complex assembled largely on damaged endothelium and not on activated platelets (see figure). Preventing platelet accumulation at the injury site had little effect on prothrombinase formation. Consistent with previous reports, the investigators showed that inhibition of platelet accumulation did not affect fibrin generation following vascular injury.9 To verify endothelial localization, the authors
1697
From www.bloodjournal.org by guest on September 11, 2016. For personal use only.
infused labeled factor Xa into mice that express green fluorescent protein (GFP) under the endothelial cell-specific Tie2 promoter. These studies confirmed that factor Xa associates with endothelial cell surfaces. Further studies showed that factor Xa localizes to areas of phosphatidylserine exposure on injured endothelium. This work defines a pathway whereby injury-dependent phosphatidylserine exposure on endothelium leads to FVa and FXa binding with subsequent fibrin generation and shows that, although platelets are able to support fibrin formation in vitro, the endothelium provides the surface for assembly of the prothrombinase complex in this in vivo model. Beyond showing how thrombi are initiated, new studies from investigators at the University of Pennsylvania reveal how platelet-rich thrombi grow and achieve their characteristic architecture. Previous work from this group demonstrated that platelet-rich thrombi consists of 2 zones: a core of tightly packed, activated platelets and a shell of loosely packed, unactivated platelets.10 In their new study, Welsh et al showed that the transport of diffusible agonists was slower in the core of the thrombus and faster in the shell.2 As an example, they showed that thrombin diffuses very little from the thrombus core, which is juxtaposed to the damaged endothelium. This arrangement sets up a transition zone in which thrombin (and other agonists) at the edge of the core activates resting platelets in the adjacent edge of the shell region (see figure). In this manner, the core grows over time. These studies demonstrate that the degree of platelet activation required to elicit granule release does not occur at the periphery of the outer shell, which is exposed to flowing blood, but rather at its core. An important correlate of this observation is that platelets accumulating in thrombi release their contents not into flowing blood but rather into the body of the thrombus. What are the consequences of releasing soluble agonists into the thrombus rather than into flowing blood? Tomaiuolo et al use a computational approach to show that the effect is profound.3 They modeled the movement of soluble agonists in a thrombus and showed that convection had very little influence on the movement of agonists released
1698
into a thrombus. Instead, the movement of these agonists was controlled by diffusion within the body of the thrombus. Modeling suggested that intrathrombus plasma velocity was ;1000-fold slower than bulk velocity in the vessel and that this “solute haven” is achieved in surprisingly small thrombi (16 platelets). The rate of diffusion is critically regulated by the packing density of platelets, which can be measured by the size of the gaps between platelets. If packing density is an important determinant of agonist diffusion in a thrombus, then modulation of packing density should have substantial effects on thrombus growth and architecture. This prediction is supported by computation studies and is validated in a mouse model in which packing density is impaired. Stalker et al studied thrombus formation and solute transport in the diYF mouse4 in which tyrosines in the b tail of aIIbb3 are mutated to phenylalanine, resulting in a defect in outside-in signaling.11 Although platelets from these mice exhibited normal fibrinogen binding and granule release, they had a defect in clot retraction that led to lower packing densities. This defect increased intrathrombus transport rates and decreased thrombin and platelet activity within the thrombus core because thrombin leached out of thrombi more quickly in diYF mice compared with wild-type controls. These results confirm that gap size, which is increased in diYF mice, is a critical determinant of solute transport and, in turn, agonist distribution and platelet activation within the thrombus. This series of articles raises important questions for the evaluation of existing antiplatelet agents and the design of novel antithrombotics. Might indications for shelltargeted antiplatelet agents (eg, aspirin, clopidogrel) differ from those for core-targeted agents (eg, hirudin, vorapaxar)? Might reagents targeting platelet packing density rather than platelet activation demonstrate better risk-benefit ratios in clinical use? In commenting on previous attempts to define blood platelets in his famous 1882 publication, Giulio Bizzozero noted that, “It is astonishing that none of the previous
investigators made use of the observation of circulating blood in living animals.”5 One hundred thirty years later, the strategy of looking closely at platelets in vivo and considering how they act as a unit, rather studying them in isolation, continues to provide information beyond that obtained by the evaluation of individual platelets. This approach will serve to improve our understanding of how thrombi form, expand, and ultimately resolve. Conflict-of-interest disclosure: The author declares no competing financial interests. n REFERENCES 1. Ivanciu L, Krishnaswamy S, Camire RM. New insights into the spatiotemporal localization of prothrombinase in vivo. Blood. 2014;124(11):1705-1714. 2. Welsh JD, Stalker TJ, Voronov R, et al. A systems approach to hemostasis: 1. The interdependence of thrombus architecture and agonist movements in the gaps between platelets. Blood. 2014;124(11):1808-1815. 3. Tomaiuolo M, Stalker TJ, Welsh JD, Diamond SL, Sinno T, Brass LF. A systems approach to hemostasis: 2. Computational analysis of molecular transport in the thrombus microenvironment. Blood. 2014;124(11): 1816-1823. 4. Stalker TJ, Welsh JD, Tomaiuolo M, et al. A systems approach to hemostasis: 3. Thrombus consolidation regulates intrathrombus solute transport and local thrombin activity. Blood. 2014;124(11):1824-1831. 5. Ribatti D, Crivellato E. Giulio Bizzozero and the discovery of platelets. Leuk Res. 2007;31(10):1339-1341. 6. Falati S, Gross P, Merrill-Skoloff G, Furie BC, Furie B. Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse. Nat Med. 2002;8(10):1175-1181. 7. Celi A, Merrill-Skoloff G, Gross P, et al. Thrombus formation: direct real-time observation and digital analysis of thrombus assembly in a living mouse by confocal and widefield intravital microscopy. J Thromb Haemost. 2003; 1(1):60-68. 8. Schlachterman A, Schuettrumpf J, Liu J-H, et al. Factor V Leiden improves in vivo hemostasis in murine hemophilia models. [published correction appears in J Thromb Haemost. 2006;4(4):925]. J Thromb Haemost. 2005; 3(12):2730-2737. 9. Vandendries ER, Hamilton JR, Coughlin SR, Furie B, Furie BC. Par4 is required for platelet thrombus propagation but not fibrin generation in a mouse model of thrombosis. Proc Natl Acad Sci USA. 2007;104(1):288-292. 10. Stalker TJ, Traxler EA, Wu J, et al. Hierarchical organization in the hemostatic response and its relationship to the platelet-signaling network. Blood. 2013;121(10): 1875-1885. 11. Law DA, DeGuzman FR, Heiser P, Ministri-Madrid K, Killeen N, Phillips DR. Integrin cytoplasmic tyrosine motif is required for outside-in alphaIIbbeta3 signalling and platelet function. Nature. 1999;401(6755):808-811. © 2014 by The American Society of Hematology
BLOOD, 11 SEPTEMBER 2014 x VOLUME 124, NUMBER 11
From www.bloodjournal.org by guest on September 11, 2016. For personal use only.
2014 124: 1697-1698 doi:10.1182/blood-2014-06-579656
Thrombus formation reimagined Robert Flaumenhaft
Updated information and services can be found at: http://www.bloodjournal.org/content/124/11/1697.full.html Articles on similar topics can be found in the following Blood collections Free Research Articles (4037 articles) Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.
