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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:728.)
© 2007 American Heart Association, Inc.

Brief Reviews

Platelet Adhesion Receptors and Their Ligands in Mouse Models of Thrombosis

Cécile V. Denis; Denisa D. Wagner

From INSERM U770 (C.V.D.), Univ Paris-Sud, Le Kremlin-Bicêtre, France, and the CBR Institute for Biomedical Research and Department of Pathology (D.D.W.), Harvard Medical School, Boston Mass.

Correspondence to Denisa D. Wagner, CBR Institute for Biomedical Research, 800 Huntington Ave, Boston, MA 02115. E-mail wagner{at}cbr.med.harvard.edu

Series Editor: David T. Eitzman
Regulation of Hemostasis and Thrombosis: Insights From Murine Models
ATVB In Focus

Previous Brief Review in this Series:

•Tollefsen DM. Heparin cofactor II modulates the response to vascular injury. 2007;27:454–460.

	Abstract

Top Abstract Introduction Tethering Activation and Firm Adhesion Aggregation and Platelet... Thrombus Stabilization Concluding Remarks and... References

 
Platelet adhesion and aggregation at sites of vascular injury are two key events in hemostasis and thrombosis. Because of exciting advances in genetic engineering, the mouse has become an important and frequently used model to unravel the molecular mechanisms underlying the multistep process leading to the formation of a stable platelet plug. In gene-targeted mice, the crucial importance of platelet adhesion receptors such as glycoprotein Ib or the IIbß3 integrin has been confirmed and further clarified. Their absence leads to highly impaired thrombus formation, independent of the model used to induce vascular injury. In contrast, the relative contribution of other receptors, such as glycoprotein VI, or of various platelet ligands may be regulated by the severity of injury, the type of vessel injured, and the signaling pathways that are generated. Murine models have also helped improve understanding of the second wave of events that leads to stabilization of the platelet aggregate. Despite the current limitations due to lack of standardization and the virtual absence of thrombosis models in diseased vessels, there is no doubt that the mouse will play a key role in the discovery and characterization of the next generation of antithrombotic agents. This review focuses on key findings about the molecular mechanisms supporting hemostasis and thrombosis that have been obtained with genetically engineered mouse models deficient in various platelet adhesion receptors and ligands. Combination of these models with sophisticated methods allowing direct visualization of platelet–vessel wall interactions after injury greatly contributed to recent advances in the field.

This review focuses on key findings about the molecular mechanisms supporting hemostasis and thrombosis, obtained with genetically engineered mouse models deficient in various platelet adhesion receptors and ligands. Combination of these models with sophisticated methods allowing direct visualization of platelet-vessel wall interactions after injury greatly contributed to recent advances in the field.

Key Words: platelets • animal models • thrombosis • adhesion • bleeding

	Introduction

Top Abstract Introduction Tethering Activation and Firm Adhesion Aggregation and Platelet... Thrombus Stabilization Concluding Remarks and... References

 
Platelets are central both to normal hemostasis, limiting blood loss after injury, and to pathological conditions, such as deep vein thrombosis and arterial thrombosis. Platelet thrombi produce devastating consequences in cardiovascular and cerebrovascular disease that are currently associated with close to 40% of all deaths in the United States.¹ In addition, thrombosis associated with malignancy is the second leading cause of mortality in cancer patients.² Platelets are thus a very important target for therapy. Historically, most of the knowledge about platelet function was derived from the diagnosis of patients suffering from bleeding and thrombotic disorders and the subsequent identification and characterization of the molecule(s) involved. In more recent years, the advent of gene targeting and transgenesis techniques in the mouse has provided a powerful in vivo setting to manipulate and study platelets. Nearly all known receptors, adhesion molecules, and many signaling molecules involved in platelet function have either been knocked out, mutated, or overexpressed in mice.³ As a consequence, mice are increasingly used as a model to explore the molecular mechanisms underlying hemorrhagic and thrombotic disorders. There is always the possibility of compensatory changes associated with gene deletions.⁴ An alternative approach, such as an inhibitor to verify the importance of a particular receptor, is recommended, because a weak phenotype of a knockout mouse could result from compensation by upregulation of another receptor with a similar role. In addition, inducible systems allowing spaciotemporal targeting of specific molecules are becoming increasingly available.

In parallel with the progress made in mouse engineering, a number of murine thrombosis models have been developed allowing direct visualization of platelet-platelet and platelet–vessel wall interactions. Detailed comparisons of existing experimental approaches used to induce arterial, venous, or microvascular thrombosis in mice have been made.^5,6 However, a brief description of the most widely used procedures appears necessary, because different results are obtained owing to the type and severity of the vascular injury, as well as the vascular bed examined. In addition, mouse genetic background, sex,⁷ age, or weight can influence the thrombotic response and the severity of injury (our unpublished observations). Most early models targeted arterial thrombosis, but more recently, venous thrombosis models are being developed that provide striking differences in the molecular players implicated. As for the type of injury, the application of ferric chloride on the outside of the vessel is one of the most commonly used techniques to generate vascular injury, in both macrovessels and microvessels.^8,9 Ferric chloride induces the formation of reactive oxygen species that lead to endothelial denudation,^10,11 resulting in prominent platelet adhesion and formation of occlusive platelet-rich thrombi (Figure 1A). Another technique involves the systemic administration of a fluorescent dye (usually rose bengal), followed by exposure of the vessel to epi-illumination at the appropriate wavelength. Photoactivation induces endothelial injury, which is limited to the focal area of epi-illumination.^12,13 Similar to ferric chloride, the damage is mediated by the formation of reactive oxygen species, and occlusive thrombi are formed. However, standardization of the vascular injury seems more difficult to achieve, because alterations ranging from normal-appearing endothelium to signs of endothelial damage (including vacuoles and eventually areas of endothelial denudation) have been reported.^6,14 One interesting feature of this model is the increased sensitivity to thrombosis of venules compared with arterioles.⁶ The third frequently used procedure is laser-induced injury, triggering very rapid formation of nonocclusive thrombi (Figure 1B).¹⁵ In this model, the intensity of the laser beam and the time of exposure will determine the severity of the lesion, ranging from activated/injured endothelium¹⁶ to endothelial denudation.¹⁷ Less common procedures such as mechanical injury induced by compression, ligation,¹⁸ or a flexible wire¹⁹ have also been described, mostly in macrovessels. Regardless of the technique used to induce endothelial damage, thrombus formation is visualized by real-time microscopy. To facilitate visualization, platelets can be fluorescently labeled either in vivo by infusion of dyes such as rhodamine 6G,²⁰ DiOC6 (dihexaoxacarbocyanine iodide),²¹ or fluorescent antibodies directed against platelet markers¹⁵ or ex vivo by application of calcein acetoxymethylester⁸ or DCF (5-carboxyfluorescein diacetate succinimidyl ester)¹⁸ before injection into recipient mice.

View larger version (29K): [in this window] [in a new window]   Figure 1. Schematic comparison of kinetics of platelet thrombus formation in the 2 most commonly used thrombosis models in mice. Time after vascular injury is indicated (min). A, Ferric chloride–induced injury leading to endothelial denudation and progressive formation of an occlusive thrombus. B, Laser-induced vascular injury leading to endothelial activation and rapid formation of a nonocclusive thrombus.

Visualization of the thrombotic process in vivo provides significantly more information than simply determining whether an occlusive thrombus can be formed in a particular genetically engineered mouse. Single-platelet adhesion, rate of thrombus growth, thrombus stability, and emboli size all can be observed and quantified in addition to measuring the time it takes for the vessel to occlude.^11,18,22 Fluorescent antibodies to various antigens present on platelets or recruited to thrombi can also be infused to localize the appearance of a particular protein.²³ Combining these powerful in vivo thrombosis models and visualization tools with the wide range of available platelet adhesion receptor/molecule mutant mice has resulted in an unprecedented opportunity to learn about the molecular players involved in specific steps of thrombus growth.

Immediately after injury, resting platelets adhere to exposed subendothelium (tethering) and become activated by strong agonists present at the site of injury, mainly collagen and the enzyme thrombin. Platelet activation leads to their firm adhesion and releases weaker agonists, ADP and thromboxane A₂, that will promote additional platelet recruitment and platelet aggregation. Drugs interfering with these agonists (Plavix and aspirin) are widely used to inhibit thrombus growth. Also, agents inhibiting IIbß3, the main platelet integrin needed for platelet aggregation, are efficacious in acute thrombotic situations. Thrombus stabilization on the vessel wall is a complex process that is supported by both platelet agonists and adhesion proteins, with some molecules having both of these functions. These different steps in platelet plug formation will now be examined, with particular emphasis on recent concepts obtained from the mouse thrombosis models.

	Tethering

Top Abstract Introduction Tethering Activation and Firm Adhesion Aggregation and Platelet... Thrombus Stabilization Concluding Remarks and... References

 
To initiate thrombus formation at the site of vessel wall damage, platelets need to be captured from the flowing blood onto the exposed subendothelium. This tethering event is mediated by a major platelet receptor that is constitutively active and expressed on resting platelets: the glycoprotein (platelet glycoprotein [GP]) Ib-IX-V complex (Figure 2).^24,25 This adhesion receptor is encoded by 4 different genes, the - and ß-subunits of GPIb, GPIX, and GPV. Deletion of the individual subunits has helped define their respective roles in the hemostatic/thrombotic response. However, targeting GPIb²⁶ or GPIbß²⁷ leads to a deficiency of the entire complex and to a phenotype similar to that of Bernard-Soulier syndrome, the human disease associated with a lack or dysfunction of the GPIb-IX-V complex.^28,29 As such, the mice display a severe bleeding phenotype, abnormal giant platelets, and severe thrombocytopenia, which preclude their use in thrombosis models. To circumvent this problem, Massberg and colleagues¹⁸ have used anti-GPIb–treated mice in a ligation-induced injury model of the carotid artery. Blocking GPIb and particularly, its ligand-binding site resulted in a virtual absence of single-platelet adhesion. In addition, recent studies in transgenic mice expressing a fusion protein made of the cytoplasmic and transmembrane portions of human GPIb (to rescue platelet generation) linked to the interleukin-4 receptor -chain³⁰ support the absolute requirement for GPIb in the process of platelet tethering and consequently in arterial thrombosis.^31,32

View larger version (41K): [in this window] [in a new window]   Figure 2. Major adhesion receptors expressed at the surface of resting platelets. On resting platelets, integrins are present in their inactive conformation. The adhesion molecules present in -granules are indicated: FG, fibrinogen; FN, fibronectin; P-Sel, P-selectin; TSP-1, thrombospondin-1, VN: vitronectin; and VWF.

The main ligand of GPIb is von Willebrand factor (VWF). VWF is a large, multimeric, plasma protein that undergoes a conformational change when bound to matrix or in pathologically high-shear conditions that permit its binding to GPIb.³³ VWF is also stored in platelet -granules from which it is released on platelet activation. The GPIb-VWF interaction is able to withstand very high shear rate conditions and is characterized by fast association and dissociation rates, allowing slow platelet translocation on the vessel wall.³⁴ Mice lacking VWF were actually the very first genetically engineered mice evaluated in a thrombosis model by intravital microscopy.⁸ In a ferric chloride–induced injury in arterioles, VWF^–/– mice exhibit delayed platelet adhesion and reduced thrombus formation (Figure 3).^8,11 However, platelets in these mice can still adhere and form thrombi, leading to the interesting observation that VWF deficiency appears less severe than the lack of functional GPIb. This suggests that GPIb can initiate platelet tethering through an interaction with ligands other than VWF.³¹ One candidate is thrombospondin-1, which was reported to bind to GPIb under high shear flow in vitro.³⁵ However, such a role for thrombospondin-1 would have to be unmasked in the absence of VWF because the absence of thrombospondin-1 does not lead to defective thrombosis in vivo.³⁶

View larger version (90K): [in this window] [in a new window]   Figure 3. Characteristics of thrombus growth in wild-type and VWF-deficient mice after ferric chloride–induced injury to arterioles and veins. Times after injury are indicated (min). A, Thrombus formation in mesenteric arterioles (diameter 100 µm and shear 1300/s). In wild-type (WT) mice, thrombi grow fast and cause vessels to occlude at the site of injury. In VWF–/– mice, although thrombi form (13 minutes), they usually stop growing at later times, leaving a small open channel (arrowheads, 29 minutes) with high shear. The thrombus appears dark, as few new platelets were recruited and platelet fluorescence was bleached after long exposure to UV light. Bar=50 µm. Reprinted with permission from Ni et al.11 B, Thrombus formation in mesenteric veins (diameter 250 to 300 µm and shear 110/s). White lines delineate the venules’ vessel walls. Thrombus growth is significantly delayed in VWF–/– mice compared with WT mice. In addition, in these mice, none of the vessels occluded during the 40-minute observation period. Bar=100 µm. This research was originally published in Blood by Chauhan et al.37

Under high-shear conditions, there is an absolute requirement for GPIb to initiate platelet adhesion; this is not the case in venous thrombosis.³⁷ In conditions of lower shear, a direct interaction between subendothelial collagens and platelet receptors (discussed next) can support adhesion.

	Activation and Firm Adhesion

Top Abstract Introduction Tethering Activation and Firm Adhesion Aggregation and Platelet... Thrombus Stabilization Concluding Remarks and... References

 
After the initial tethering and translocation of platelets on the exposed subendothelium, a number of agonists can initiate signaling events that will lead to platelet activation, followed by firm adhesion. A very important aspect of platelet activation is the transition of a low-affinity binding state of platelet integrins to a high-affinity state. The activation of platelets by adhesion to exposed collagen will be discussed first. Two collagen receptors have been identified on the platelet surface: the integrin 2ß1 and a member of the immunoglobulin superfamily, GPVI (Figure 2).³⁸ Defining the respective roles of these 2 receptors generated intense debate, and mice deficient in these receptors helped to clarify the issues. Two groups generated 2-deficient mice^39,40 and reported the formation of occlusive thrombi in thrombosis models^41,42 (the Table), although occlusion time was delayed in one of those studies.^41,42 This result is in accordance with the observation that 2-deficient mice display normal bleeding times and supports the notion that 2ß1 is not required for platelet adhesion in vivo. Similarly, a Cre-/loxP–mediated loss of the ß1-integrin subunit in mouse platelets, resulting in the concomitant loss of 2ß1, 5ß1, and 6ß1, led to normal thrombus formation after ligation-induced injury of the carotid artery.⁴¹ The second collagen receptor, GPVI, is associated with the signaling adapter Fc receptor- (FcR) chain. For a long time, GPVI was considered a very attractive target for the development of new antithrombotic drugs.⁴³ Using antibody-depleted GPVI-deficient mice in ferric chloride–induced or mechanical vascular injury, both exposing subendothelial collagen, Massberg et al¹⁸ reported a dramatic reduction of platelet tethering, translocation, and firm adhesion as well as a delay in occlusion time (the Table). Because GPVI-depleted mice displayed only slightly prolonged bleeding times, that observation raised the interesting possibility that GPVI could be playing a major role in pathological arterial thrombosis but not in physiological hemostasis.⁴³ However, later studies have not confirmed this role of GPVI as an adhesion receptor, and it now appears that the main function of the GPVI-collagen interaction is the generation of intracellular signals promoting activation of integrins such as 2ß1 and IIbß3. Indeed, the lack of GPVI can be compensated for by direct integrin activation by Mn²⁺ to restore platelet adhesion to collagen in vitro.⁴⁴ Is GPVI necessary for this activation step in vivo? GPVI-deficient mice that have recently been studied in ferric chloride–induced injury in the carotid artery exhibited a very variable phenotype, with some mice behaving like their wild-type littermates while others presented a markedly abnormal thrombotic response.³² The reason for such a difference is not completely understood but could be related to the extent of vascular injury and whether another activation pathway could compensate for the loss of GPVI. Indeed, in different types of thrombosis models, it was shown that mice deficient in FcR, which are also deficient in GPVI, displayed an abnormal thrombotic response only in models leading to important collagen exposure (Figure 1), such as ferric chloride–induced injury.¹⁶ In the laser-induced injury model, however, collagen exposure was undetectable and thrombus formation was normal in FcR-deficient mice. It seems that platelet activation is not dependent on GPVI after laser injury but rather on thrombin, because higher levels of thrombus-associated tissue factor (TF) could be detected in this model.¹⁶ This possibility was further investigated by Mangin et al,²¹ who compared three models of arterial thrombosis and found that loss of GPVI/FcR led to a minimal defect in thrombus formation but to a severe defect when additional treatment with thrombin inhibitors was performed. This study highlights the redundancy of the system by which platelets can become activated and demonstrates the functional overlap between thrombin and GPVI/FcR. At the same time, it questions the possibility of using GPVI as a potential antithrombotic target because to obtain good protection against thrombosis, it would be necessary to target both GPVI and thrombin, a combination that significantly prolongs bleeding time.¹⁵

View this table: [in this window] [in a new window]   Thrombotic Phenotype of Mice With Defects in Platelet Adhesion Receptors and Their Ligands

View this table: [in this window] [in a new window]   Continued

Besides GPVI, a number of other important activation receptors are present on the surface of platelets, such as members of the protease-activated receptor (PAR) family, PAR-3 and PAR-4, the murine thrombin receptors.⁴⁵ Thrombin is rapidly generated at the site of vascular injury and is considered the most potent platelet activator, leading to shape change, integrin activation, and granule secretion. Thrombin inhibition reduces thrombus formation in thrombosis models.^16,21,45,46 Later, a second wave of mediators can potentiate the activation. The best known is ADP released from platelet dense granules and binding to P2Y₁ and P2Y₁₂ receptors on the platelet surface.⁴⁷ Platelet receptors for agonists such as thromboxane A₂⁴⁸, epinephrine,⁴⁹ ATP,⁵⁰ or even leptin⁵¹ have also been identified and their in vivo functions tested in knockout mice. Most of these receptors are coupled to heterotrimeric G proteins that function as intracellular signaling molecules. Mice deficient in a number of these G proteins have also been generated.⁵² The platelet signaling pathways were recently reviewed.^3,53 An important notion is that platelet activation is a dynamic process in which the different receptors and signaling pathways are linked to one another. Furthermore, the distinction is not always perfectly clear between adhesion and activation receptors. For example, on ligand binding, the GPIb-V-IX complex has been shown to mediate intracellular signaling, leading to platelet activation.⁵⁴ In addition, under conditions of low thrombin concentrations, GPIb can also participate in the activation process by using its capacity to act as a thrombin receptor,^55,56 whereas integrin engagement also produces activating signals within the platelets.⁵⁷ Independent of the activation pathway used by the platelets, the common resulting event is the activation of platelet integrins. The IIbß3 integrin is the major platelet integrin used for both firm adhesion and platelet aggregation, the next step in the series of events leading to thrombus formation.

	Aggregation and Platelet Recruitment

Top Abstract Introduction Tethering Activation and Firm Adhesion Aggregation and Platelet... Thrombus Stabilization Concluding Remarks and... References

 
Once firmly adherent on the vessel wall, platelets start to spread and release the contents of their granules, providing an adhesive surface to recruit new platelets (Figure 4). Recently, experimental in vitro and in vivo evidence showed that the recruitment is primarily mediated by GPIb on the incoming platelet.^31,58,59 Thus, the GPIb complex is crucial in both the initial step of platelet adhesion to the injured vessel wall and in the recruitment of new platelets to the growing thrombus. The activated platelets release ADP and thromboxane A₂, further promoting activation.⁶⁰ This self-amplifying process will lead to the formation of the platelet plug and the arrest of bleeding. The key adhesion molecule mediating the aggregation step is IIbß3, which is present in platelets, not only on the plasma membrane but also in -granules, providing an extra available pool on platelet activation.⁶¹ Absence or dysfunction of IIbß3 in humans leads to the bleeding syndrome known as Glanzmann thrombasthenia.⁶² Mice with a targeted deletion of the ß3-subunit represent a very good model of this disease, with prolonged bleeding time, gastrointestinal hemorrhage, abnormal platelet aggregation, and clot retraction.⁶³ Blocking IIbß3 in mice, either through gene targeting or by using IIbß3 antagonists, has been shown to block thrombus formation irrespective of the thrombosis model, emphasizing the crucial importance of this receptor (the Table).^11,21,41,64

View larger version (77K): [in this window] [in a new window]   Figure 4. Schematic representation of the major platelet adhesion receptors and ligands involved in the formation of a stable arteriolar thrombus in mice. The electron microscopy photograph represents a platelet thrombus formed on subendothelium. Inserts provide more detailed information about the molecular mechanisms involved at the different interfaces of the thrombus (platelet–vessel wall or platelet-platelet). Depending on the ligand involved, different collagen sequences have been identified, as indicated in the figure.105 FG indicates fibrinogen; FN, fibronectin; P-Sel, P-selectin; TF, tissue factor; VWF; and ?, unknown ligand.

A number of ligands of IIbß3 have been identified through in vitro experimentation, such as fibrinogen, VWF, and fibronectin (FN). Because fibrinogen was thought to be the major IIbß3 ligand involved in in vitro platelet aggregation, the observation that thrombus formation was not abolished in fibrinogen-deficient mice came as a surprise.¹¹ However, the thrombi in fibrinogen-deficient mice were unstable and were seen to embolize frequently, leading to occlusion downstream from the injury site. The thrombi appeared to be poorly attached to the vessel wall, suggesting that the primary role for fibrinogen at high shear might not be to mediate platelet homotypic interactions but rather to form fibrin to stabilize the growing thrombus. Further studies in mice expressing a truncated form of fibrinogen (Fg5), which retains the ability to convert to fibrin but is unable to bind to IIbß3, have helped distinguish between these 2 functions of fibrinogen. Indeed, although thrombi formed in Fg5 mice also embolized, the emboli were relatively small and were released from the top of the thrombi rather than by fracture at the vessel wall.⁶⁵ In a carotid artery injury model, embolization in the absence of fibrinogen produced a significant delay in the occlusion of these larger vessels.²² Furthermore, Fg5 mice developed only small thrombi that appeared pacified (no longer adhesive to new platelets) by a fibrin core. The differences could be explained by the different size and hemodynamic properties of the injured vessels examined in those studies. Taken together, these observations support the concept that fibrin(ogen) contributes to thrombus stability by two mechanisms: (1) fibrin formation anchoring the thrombi to the vessel wall and (2) IIbß3 binding, together with other ligands, helping to stably anchor individual platelets to the thrombus.

Among other adhesive proteins involved in platelet aggregation, VWF was shown to play an essential role in occlusive thrombi formation at both venous³⁷ and arterial¹¹ shear rates (Figure 3). Indeed, at the apex of the developing thrombus, where very rapid flow is encountered, the absence of VWF is particularly deleterious, preventing additional growth of the thrombus and subsequent occlusion.¹¹ The importance of VWF in regulating thrombus growth was further underlined in the mouse model of thrombotic thrombocytopenic purpura lacking the metalloprotease ADAMTS13 (A Disintegrin And Metalloproteinase with a ThromboSpondin type 1 motif, member 13),⁶⁶ which proteolytically reduces the size of VWF multimers in plasma, thus lowering their affinity for platelets. In mesenteric arterioles of ADAMTS13-deficent mice, with the ferric chloride injury model, thrombi grew much faster and occlusion occurred significantly earlier than in wild-type mice.⁶⁷ Infusion of recombinant ADAMTS13 into wild-type mice, on the other hand, delayed arteriolar occlusion, with several mice never producing an occlusive thrombus.⁶⁷

An unexpected observation was made when arterial thrombosis was studied in mice lacking both of the principal adhesion molecules, VWF and fibrinogen. Delayed platelet adhesion and aggregation were observed in these mice, and even when the thrombi were fragile and unstable, vessel occlusion was not uncommon.¹¹ These observations initiated the search for additional mechanisms that could promote platelet aggregation. The search was first narrowed to IIbß3 ligands, because ß3-deficient mice were unable to form thrombi in the same thrombosis model. A number of candidate genes were considered. As mentioned earlier, thrombospondin-1–knockout mice do not exhibit thrombosis defects,³⁶ and the plasma concentration of this protein is negligible. However, thrombospondin-1 is released from platelet -granules on activation. Therefore, one cannot rule out a potential compensatory role for thrombospondin-1 in the absence of both VWF and fibrinogen. Vitronectin was also considered, but for the moment, its role in thrombosis is highly controversial^68–71 (the Table). Another interesting candidate to mediate VWF/fibrinogen-independent thrombus formation was FN, partially owing to the fact that its content was elevated in the platelet -granules of VWF/fibrinogen-deficient mice through import by IIbß3.^11,65 Because ablation of the FN gene causes embryonic lethality, a conditional knockout mouse model with reduced plasma FN levels (<2% of wild type)⁷² was used to address the role of this protein in thrombosis. In arterioles, ferric chloride–induced injury led to highly defective thrombus formation, with small aggregates constantly detaching from the growing thrombi and resulting in a much-delayed vessel occlusion.³⁶ Interestingly, a similar phenotype of thrombosis was observed in FN-heterozygous mice, showing that even a 50% reduction in FN levels drastically affects arterial thrombosis.⁷³ However, no defect was found at venous shear,⁷³ indicating that the importance of FN is specific to the arterial side of the circulation. Although the studies clearly demonstrate a role for FN in platelet thrombus stability in arteries, it is not yet clear which mechanism is involved and which combination of the 3 FN platelet receptors, IIbß3, vß3, and 5ß1, participates in this process. FN could also stabilize the fibrin clot and thus enhance platelet cohesion.⁷⁴ Clearly, the aforementioned studies document the existence of several integrin ligands and point to a number of alternative pathways involved in platelet aggregation. This conclusion is further supported by a recent report demonstrating that both plasma and platelet granule proteins contribute to VWF/fibrinogen-independent platelet aggregation, thus emphasizing the complexity of the system⁷⁵ (Figure 4). Under normal conditions (wild-type mice), the relative importance of these different proteins is difficult to assess. However, the data available so far point to a role for VWF and FN in normal thrombus formation at arterial shear.

Besides the "usual suspects" of proteins involved in platelet aggregation discussed so far, recently a new IIbß3 ligand has been identified. This molecule, CD40L (CD154), known to participate in immune responses and being a member of the tumor necrosis factor family, is released on platelet activation.⁷⁶ In mice, the absence of CD40L was shown to affect the stability of arterial thrombi, leading to delayed vessel occlusion.⁷⁷ Recombinant soluble CD40L via its KGD-integrin binding sequence could restore thrombus stability.⁷⁷ It is interesting to note that, similar to mice with reduced FN levels, CD40L-deficient mice did not present prolonged bleeding times. Further studies indicated that CD40L was inducing platelet stimulation through IIbß3-dependent outside-in signaling.⁷⁸ This delayed wave of platelet activation by protein ligands of platelet receptors represents a rather new and exciting field in thrombosis and will be discussed in the next section.

	Thrombus Stabilization

Top Abstract Introduction Tethering Activation and Firm Adhesion Aggregation and Platelet... Thrombus Stabilization Concluding Remarks and... References

 
In the last few years, it has become apparent that the primary activation and aggregation step is followed by a second wave of activation signals meant to prevent platelet aggregates from falling apart. Clot retraction is also an illustration of this process. Indeed, platelet-mediated clot retraction is necessary for the consolidation of a platelet thrombus by making it less susceptible to fibrinolysis. This event is virtually absent in patients or in mice lacking IIbß3, suggesting a major role for this receptor in stabilization of the thrombus. It is now known that platelet-platelet contacts occurring during aggregation lead to outside-in signaling through IIbß3.⁷⁹ Phosphorylation of tyrosine residues on the cytoplasmic tail of the ß3-integrin subunit is part of the signaling event induced by ligand occupancy.⁸⁰ The defect in stabilization of platelet thrombi in CD40L-deficient mice is a good example of the importance of this process.^77,78 The thrombi formed in the arterioles of these mice looked lacelike rather than compacted, as seen in wild-type mice.⁷⁷ CD40L appears to be a part of an autocrine loop, because it is released when platelets are activated by primary platelet agonists and it then further enhances platelet activation (Figure 4). The growth arrest–specific gene 6 (Gas6)/Axl-Tyro3-Mer receptors represent another ligand-receptor pair with a similar effect. Gas6 is a vitamin K–dependent protein that is stored in platelet -granules.⁸¹ When platelets become activated, Gas6 is released and binds to its receptors on the platelet surface, reinforcing IIbß3 signaling through cross-talk between the receptors.⁸² Targeting Gas6 in mice results in the formation of much smaller thrombi both in veins and in arteries compared with those observed in wild-type mice.⁸¹ Comparable results were obtained with Gas6 receptor–deficient mice⁸³ and with mice injected with blocking antibodies against Gas6 or the Gas6 receptor.⁸⁴ Transmembrane proteins interacting with IIbß3 have also been implicated in thrombus stabilization. The tetraspanin family members, CD151⁸⁵ and TSSC6,⁸⁶ are able to regulate the outside-in IIbß3 signaling. TSSC6-deficient mice display increased embolization and impaired thrombus stabilization after ferric chloride–induced injury.⁸⁶

As previously mentioned, the persistence of a stable hemostatic plug requires close contacts between platelets. Indeed, contact-dependent signaling requires platelet-platelet interactions of sufficient duration.⁸⁷ Such a concept has been described for the lymphocyte synapse induced during the immune response, generating a proimmune environment. By analogy, the hypothesis of a "platelet synapse," whereby boundaries between adjacent platelets would be populated by signaling and adhesion molecules, thus creating a prothrombotic environment, has been proposed.^87,88 A growing number of molecules have now been identified that play a role in these late events of platelet activation. Among these are ephrin/Eph receptor kinases.⁸⁹ Both the ligand (ephrin) and the receptor (Eph kinase) are cell surface molecules (either glycosylphosphatidylinositol anchored or with a single transmembrane domain) that bind to each other on opposing platelets, causing signaling in both cells. So far, the role of ephrin/Eph kinases in thrombus stabilization has only been studied in vitro.⁹⁰ Other aggregation-induced coreceptors include PEAR1 (Platelet Endothelial Aggregation Receptor 1), identified through a proteomics approach,⁹¹ as well as members of the SLAM (Signaling Lymphocyte Activation Molecule) family of homophilic adhesion receptors such as CD84 and CD150.⁷ In vivo analysis of SLAM-deficient mice has revealed normal bleeding times but impaired thrombosis after ferric chloride injury in female but not in male mice. These mice displayed delayed appearance of the first thrombus and occlusion of the vessel with increased embolization.⁷ In addition, semaphorin 4D (CD100) and its receptors CD72 and plexin-B1 have been recently identified on platelets, and mice deficient in semaphorin 4D exhibit delayed arterial occlusion after arteriolar injury in vivo.⁹²

The final pathway contributing to thrombus stability is the formation of the fibrin network deriving from the coagulation cascade. In a laser-injury thrombosis model (Figure 1B), it was recently demonstrated that normal fibrin formation is dependent on the recruitment of blood-borne (microparticle) TF to the thrombus, with only a minimal contribution of vessel wall TF.⁹³ In cremaster muscle arterioles of mice expressing low levels of blood-borne TF, smaller thrombi were formed than in wild-type mice. The microparticles express P-selectin glycoprotein ligand 1 (PSGL-1) and are recruited into the thrombus via binding to P-selectin expressed on the activated platelets.⁹⁴ Mice deficient in either P-selectin or PSGL-1 develop platelet thrombi containing little TF or fibrin. This result complements the observation that high levels of soluble P-selectin are associated with a procoagulant state.⁹⁵ In addition, engagement of P-selectin with its receptor PSGL-1 on leukocytes leads to the generation of microparticles, which can correct hemostasis in a murine model of hemophilia A.⁹⁶ The relative importance of blood-borne versus vessel wall TF was, however, questioned when Day et al⁹⁷ found that thrombus formation was driven primarily by vessel wall TF in photochemically induced injury in the carotid artery. Therefore, it appears that the importance of blood-borne TF for local thrombin generation in the thrombus depends initially on the extent of vessel injury,⁹⁸ with a large injury producing enough TF from the ruptured tissue. Later, the hematopoietic cell–derived TF should help to promote thrombus stability until healing is completed. Another source of TF may originate from platelets themselves. Recently, platelets were shown to express TF pre-mRNA, which can be spliced into mature mRNA on platelet activation, leading to the production of bioactive TF protein.⁹⁹ However, the role of this platelet-derived TF in thrombus formation still needs to be addressed in vivo.

	Concluding Remarks and Perspectives

Top Abstract Introduction Tethering Activation and Firm Adhesion Aggregation and Platelet... Thrombus Stabilization Concluding Remarks and... References

 
The development of genetically engineered mice, along with sophisticated in vivo analysis methods, has impressively augmented our knowledge of the molecular mechanisms supporting hemostasis and thrombosis. However, great care should be taken in selecting a particular thrombosis model, and the limitations of each model need to be recognized. As illustrated in this review, different results may be obtained according to the extent of injury or the vascular bed targeted. Interestingly, there may be specificity in the involvement of particular adhesion molecules, depending on whether thrombosis occurs in the venous or arterial circulation (the Table). Despite the fact that most studies to date have addressed arterial thrombosis and very few have compared the importance of specific molecules in venous versus arterial thrombosis, differences have already appeared. Whereas VWF seems to be equally implicated in veins and arteries (Figure 3), GPIb and FN are both more important in the arterial side of the circulation. Such differences may allow production of inhibitors specific to the venous or arterial setting.

In the next few years, the greatest challenge will be to develop mouse models of pathological thrombosis that mimic human vascular disease. Although molecular mechanisms responsible for thrombus formation in the microcirculation of a healthy mouse may be similar to those of healthy humans and the knockout mouse provides satisfactory models for common genetic diseases involving thrombosis, mouse models of atherothrombosis are lacking. Mice do not develop complex atherosclerotic lesions without genetic manipulation to increase their plasma cholesterol levels. In one such mouse deficient in apolipoprotein E, indirect evidences of atherosclerotic plaque ruptures were noticed during the past several years,¹⁰⁰ but only recently could such rupture be induced experimentally.^101,102 Apolipoprotein E–deficient mice crossed with mice deficient in adhesion molecules might therefore help to identify the important players in plaque rupture–induced thrombosis. Emerging techniques providing deeper tissue penetration, such as near-infrared imaging¹⁰³ or multiphoton microscopy, could be utilized to visualize the plaque rupture process. Mouse models of stroke¹⁰⁴ that better reflect human pathology also need to be perfected. The effects of middle cerebral artery occlusion in a healthy mouse are likely quite different from a stroke resulting from an embolus after atherosclerotic plaque rupture. Also, it might be difficult to develop new therapies for thrombus dissolution in a stroke model wherein the primary occlusion is caused by a suture, as is done routinely in mice.

Although the relevance of mouse studies to human pathology may not always be straightforward, mice have already been proven to provide an excellent model to study thrombosis and with continuing improvements, will remain a practical tool for identifying and validating novel targets for therapeutic intervention.

	Acknowledgments

 
We thank Wolfgang Bergmeier, Peter Lenting, and the reviewers for helpful criticism of the manuscript; the Department of Clinical Chemistry and Hematology, UMC Utrecht (The Netherlands) for providing electron microscopy images; and Lesley Cowan for help with the preparation of the manuscript.

Sources of Funding

The work presented from our laboratories was funded by grants from the National Heart, Lung, and Blood Institutes of the National Institutes of Health R37 HL41002 and P01 HL56949 (to D.D.W.) and Institut National de la Santé et de la Recherche Médicale (to C.V.D.).

Disclosures

None.

	Footnotes

 
Original received November 30, 2006; final version accepted January 23, 2007.

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