Bruce Furie, 840 Memorial Drive, Cambridge, MA, 02139, USA Tel.: +1 617 667 0620; Fax: +1 617 975 5505; e-mail: firstname.lastname@example.org
Summary. Thrombus formation, including platelet adhesion, activation, secretion and aggregation as well as tissue factor-initiated thrombin generation and fibrin formation, has been studied in the past using in vitro systems, often with isolated components. Given the complexity of hemostasis and thrombosis, many of the concepts that have been developed to explain these processes are being revisited by studying thrombus formation in live animals using intravital microscopy and genetically altered mice. Although much of the dogma that has evolved has been confirmed by in vivo studies of thrombus formation, there have also been conflicts between old concepts and new direct observations. In vivo studies of the initiation of thrombus formation, platelet accumulation and thrombin generation have provided evidence for the participation of novel proteins and identified new pathways and mechanisms.
The hemostatic system is a host defense mechanism that preserves the integrity of the high pressure closed circulatory system in mammals. This system is, by necessity, highly complex. It involves a vessel wall, tiled with endothelial cells, that maintains an inert, inactive surface that supports blood flow without interaction of blood cell components with the blood vessel. It requires soluble plasma proteins, some of which participate in blood coagulation or as regulators of blood coagulation. Cellular components of blood include red blood cells, platelets and leukocytes, including granulocytes, monocytes and lymphocytes. Microparticles, derived from leukocytes and platelets, also circulate in the blood, although information about their functional role is limited. These components play a primary role in various forms of host defense, but may participate directly in the hemostatic process. Under normal conditions they circulate in the blood as inactive and inert constituents. Upon tissue injury, the system is activated. Tissue disruption of the endothelium leads to exposure of the subendothelial matrix. Alternatively, endothelial cells are activated, with the expression of receptors on the plasma membrane, exocytosis of the Weibel–Palade bodies and initiation of internal biochemical processes. Changes in the plasma membrane alter the phospholipid composition presented to the flowing blood. With the expression of tissue factor, the zymogens of the blood clotting proteins are converted sequentially to enzymes, and thrombin is rapidly formed. Thrombus formation is initiated. This system is precisely regulated, and critical regulatory pathways have been identified. When the activated hemostatic system overwhelms the normal regulatory controls that contain and localize thrombus formation to the area of injury, pathologic thrombosis occurs.
The protein and cellular components involved in hemostasis and thrombosis have been extensively studied, often in isolation, using techniques common to the study of biochemical processes, cell biology and molecular biology. Given the large number of participants in this complex process, it has become necessary to study hemostasis and thrombosis in an intact live animal in the presence of an active vessel wall, flowing blood, calcium and all of the components of blood. It is now possible to study thrombus formation in a live mouse given the convergence of novel technologies involving high speed digital imaging, robust computer-based image analysis and genetically modified mice. Our intravital microscopy system, described previously [1,2], but continuously evolving to allow faster data acquisition, increased data storage, and improved image resolution, is an example of the intravital microscopy technologies applied to the study of thrombus formation in live animals.
The study of thrombus formation in vivo
Monitoring spontaneous thrombus formation during intravital microscopy experiments is impractical using current mouse models, so thrombus formation is induced during the course of the experiment. A number of mouse models of thrombosis have been studied, and they each have advantages and disadvantages. Mechanical disruption of the vessel wall, photo-oxidation of the endothelium using Rose Bengal dye, vessel ligation, oxidation and vessel wall denudement of endothelium with ferric chloride [3–5] and laser-induced injury of the vessel wall [1,6] are among the techniques employed. They each probably mimic a different type of thrombus pathophysiology that can occur spontaneously in humans. Because of its high degree of spatial and temporal resolution, we have focused our work on laser-induced vessel wall injury.
New insights from in vivo studies of thrombus formation
The ability to study the biochemistry and cell biology of complex systems in living animals has allowed re-examination of the concepts that provide a foundation for our understanding of thrombus formation. These foundations include concepts of adhesion, aggregation, activation and secretion events in platelets, evaluated in vitro, but synthesized into a unifying conceptual framework to explain platelet thrombus formation. Similarly, the biochemistry of the blood coagulation cascade, starting with initiation by tissue factor, the formation of protein complexes on membrane surfaces, and culminating in the generation of thrombin and the formation of a fibrin clot, has been extensively analyzed in vitro. Although many of the constructs developed from in vitro studies are plausible, as a first approximation, intravital studies of thrombus formation have established some important features of thrombus formation that require reconsideration of pathways, mechanisms and protein participants.
Contact phase of blood coagulation
Although the intrinsic pathway describes the biochemical events, from factor (F) XII to thrombin generation, that occur in vitro, a physiologic role for this pathway was dismissed previously as patients with FXII deficiency do not have a hemostatic disorder and hemostatic abnormalities are only variably observed in patients with severe FXI deficiency. Given the ability of FVIIa/tissue factor to activate FIX directly , the in vivo pathway of blood coagulation has been thought to be initiated by tissue factor and involve the activation of FIX and the generation of the tenase complex, FIXa/FVIIIa. However, in an in vivo model of thrombus formation, Renne et al.  demonstrated that mice lacking FXII exhibit defective thrombus formation. Similar results were observed with mice lacking FXI . Current speculation is that the pathways for hemostasis may differ from the pathways of thrombosis, as it is possible to attenuate thrombosis without inhibiting hemostasis by targetting FXII for inhibition . This concept offers an attractive strategy for developing novel and effective antithrombotics that would eliminate the bleeding risk associated with such agents.
Blood-borne tissue factor
Tissue factor antigen can be detected in whole blood, contradicting the dogma that tissue factor is constitutively maintained in a compartment separated from flowing blood [11–13]. However, no activity can be detected . Nemerson et al.  discovered blood-borne tissue factor incorporation into experimental thrombi on pig arterial media or collagen-coated glass slides exposed to flowing native human blood in an in vitro perfusion system. These data challenged the existing paradigm that tissue factor is not exposed to blood prior to vascular injury, and raised the possibility that microparticles are a source of blood-borne tissue factor that is involved in thrombus propagation at the site of vascular injury. Tissue factor in blood may be encrypted so as not to cause thrombosis in the absence of specific stimuli. Recent reports have proposed that oxidation of a redox-sensitive disulfide bond in tissue factor may trigger the conversion of tissue factor to its active form [16,17]. Alternatively, the concentration of tissue factor may be below the threshold for activation of blood coagulation, until tissue factor is concentrated at a site of vascular injury. During in vivo experiments, we observed tissue factor antigen accumulation into the growing thrombus in a live mouse, and this tissue factor was active, as fibrin, the end product of tissue factor-initiated blood coagulation, was generated . This tissue factor is associated with microparticles . Tissue factor-bearing microparticles circulate constitutively in normal blood and accumulate in the developing thrombus after vessel wall injury in a mechanism that is P-selectin and PSGL-1-dependent . In fact the original observation linking P-selectin and fibrin generation emerged from study of a baboon model of thrombosis . Both vessel wall and microparticle tissue factor participate in fibrin generation and platelet activation in the laser-induced thrombosis model, and microparticle tissue factor appears to play a dominant role in fibrin generation and thrombus propagation. Although leukocyte tissue factor and microparticle tissue factor in the circulation could potentially be delivered to the developing thrombus, initial blood-borne tissue factor is derived from microparticles . In chimeric mice deficient in tissue factor in the vessel wall but containing hematopoietic, blood-borne tissue factor, tissue factor and fibrin accumulate in the developing thrombus . This is proof of the presence of blood-borne tissue factor in vivo. In thrombosis models in which there is no vessel wall tissue factor , where vessel wall injury causes vessel wall tissue factor to dominate , or where there is no blood flow, thus eliminating the deposition of microparticles , the balance between the contribution of vessel wall tissue factor and microparticle tissue factor can be altered, giving varying results. Different pathologies associated with thrombosis may reflect varying contributions of tissue factor from the vessel wall and from blood microparticles.
Interrelationship of platelet thrombus formation and fibrin clot formation
The classic teaching has been that platelets adhere to the injured vessel wall, form platelet aggregates and generate the platelet plug. Subsequently, the platelet plug is stabilized by the formation of a fibrin meshwork. However, it is now clear that platelet activation and platelet thrombus formation are events that are temporally and spatially intertwined with thrombin generation and fibrin clot propagation . Using multichannel fluorescence intravital microscopy to image thrombus formation in real time, we demonstrated that the platelet thrombus forms as fibrin spreads through the developing thrombus (Fig. 1).
Protein complex formation on membrane surfaces
Thrombin generation requires several surface-mediated enzymatic reactions during blood coagulation. The tenase complex, with FIXa in complex with FVIIIa on membrane surfaces, activates FX. The prothrombinase complex, with FXa in complex with FVa on membrane surfaces, activates prothrombin. The cell membranes that support these reactions in vivo are unknown but the activated platelet membrane has been assumed to play the dominant physiological role. In in vitro systems using purified phospholipid vesicles, the importance of anionic phospholipids, particularly phosphatidylserine, has been known for many decades. On cell membranes, phosphatidylserine is sequestered in the inner leaflet of the lipid bilayer and is only translocated to the outer leaflet upon cell activation. Many blood cells, including platelets and endothelial cells, undergo this transformation and are able to support membrane-dependent blood coagulation in vitro. However, this construct may need to be reconsidered. Although Par4 null mice do not generate a significant platelet thrombus as these platelets are refractory to thrombin-induced activation following laser injury of the vessel wall, normal amounts of fibrin are generated around the injury . As fibrin formation is thrombin-dependent and as thrombin generation requires membrane surfaces, it begs the question of whether platelet membranes are really involved in this process. Minute quantities of activated platelets may be sufficient to support thrombin generation. Alternatively, other cell surfaces, such as endothelial cell membranes or microparticle membranes, may be physiologically relevant. The dogma that activated platelets provide the membrane surface for thrombin generation needs to be questioned and the critical membrane surfaces in vivo determined.
Thrombin generation in the developing thrombus
The blood clotting cascade culminates with the generation of thrombin. Using a fluorogenic peptide substrate based upon fibrinogen, thrombin activity can be visualized within and throughout the growing thrombus in a live mouse . Thrombin activity is expressed following vessel wall injury, and the kinetics of its expression correlate with platelet thrombus development. However, after reaching peak activity, thrombin activity decays rapidly. In vivo results indicate that thrombin activity is distributed throughout the platelet thrombus, and not just at the thrombus–blood interface. Thrombin activity is attenuated following peak platelet thrombus formation, although it is not clear whether this is due to decreased thrombin production or inhibition of thrombin. These observations indicate that the arterial thrombus is porous, allowing the flow of substrate through the thrombus.
Platelet–vessel wall interaction
Platelet–vessel wall interaction is mediated by several important adhesive molecules. At low shear rates (0–1000 s−1), this interaction is highly dependent upon fibrinogen. At intermediate shear rates (1000–10 000 s−1), both fibrinogen and von Willebrand factor (VWF) play critical roles. At high shear rates in excess of 10 000 s−1, VWF dominates as the critical adhesive molecule. Following vascular injury of arterioles in the mesentery microcirculation, Maxwell et al.  showed discoid platelets translocating over bound platelets within a thrombus via a sliding motion. These platelets form thin membrane tethers to the superficial layers of the thrombus, and both fibrinogen and VWF contribute to these tethers at physiologically relevant shear rates.
We monitored calcium mobilization as a reporter of platelet activation during thrombus formation in live mice to distinguish platelet activation from platelet accumulation. Fura 2-loaded donor platelets (green) were infused into recipient mice, and the formation of the platelet thrombus monitored. Taking advantage of the changes in the fluorescence properties of this flurochrome when it binds to calcium, we imaged calcium mobilization directly in platelets (yellow) as a marker of platelet activation in circulating platelets associated with the developing thrombus. In wild-type mice, the Fura 2-loaded platelets adhered and accumulated at the site of injury. Some of those platelets mobilized intracellular calcium whereas many remained in their resting state (Fig. 2A). When Fura 2-loaded platelets were treated with the calcium chelator BAPTA-AM prior to infusion into a donor mouse, calcium mobilization was inhibited. However, BAPTA-AM-treated Fura 2-loaded platelets transiently bound to the developing thrombus. These results indicate that non-activated platelets become part of the developing thrombus and that platelets in which calcium mobilization is inhibited can also become associated with the developing thrombus. Furthermore, the dynamics of thrombus formation indicate the transient and reversible nature of non-activated platelet interaction with the thrombus. The absence of GPIb significantly impairs thrombus formation, emphasizing its importance in this process .
The role of VWF in platelet accumulation has been studied using in vivo models of thrombosis. Denis et al.  and Ni et al.  have shown in the ferric chloride thrombosis model that thrombi form even in mice lacking VWF. However, the thrombi are smaller and fewer thrombi are occlusive. We have similarly determined that, in the absence of VWF, platelet accumulation was attenuated during thrombus formation . These systems at intermediate shear rates are dependent, at least in part, on VWF.
Two pathways to platelet activation: collagen-mediated and thrombin-mediated
A compelling argument is evolving to indicate that there are two separate pathways to platelet activation. In one pathway, the subendothelial matrix is exposed following injury of the endothelium, with exposure of collagen within the subendothelial matrix to flowing blood. This pathway has been extensively studied in vitro and in vivo, and has emphasized the importance of the GPIb/V/IX complex, VWF and glycoprotein VI(GPVI). Dubois et al.  compared platelet thrombus formation in wild-type and in FcRγ null mice deficient in GPVI using the ferric chloride model for initiating thrombus formation. Ferric chloride causes major disruption of the endothelium , and leads to the exposure of collagen in the subendothelial matrix . Platelets accumulate and co-localize with collagen in wild-type mice. In an injury model that is dominated by collagen-induced platelet activation  or a model where thrombin is inhibited , FcRγ null mice lacking GPVI show no platelet accumulation and mice lacking GPVI show markedly attenuated platelet accumulation. This emphasizes the requirement for GPVI on the platelet surface and an important role for VWF in platelet accumulation in the collagen-initiated pathway.
In contrast to the ferric chloride thrombosis model, our laser-induced vessel wall injury model is dominated by the tissue factor-mediated generation of thrombin. We have repeatedly observed the rapid appearance (1–2 s) of tissue factor antigen on the vessel wall-thrombus interface in arterioles. This thrombosis model, which has features similar to those characteristic of inflammation, does not disrupt the subendothelial matrix, and specifically does not involve the exposure of collagen, in contrast to ferric chloride injury . Under the conditions that we employ, the tissue factor-mediated pathway for thrombin generation is the major mechanism for platelet activation. Inhibition of thrombin activity using hirudin blocks in vivo platelet thrombus formation as well as fibrin generation. Furthermore, Par4 null mice lacking this thrombin receptor demonstrate minimal platelet thrombus formation following laser-induced injury in vivo . However, FcRY null mice undergo normal thrombus formation in this model . These results indicate the importance of thrombin-mediated platelet activation in this thrombosis model.
Previous in vitro studies have described two calcium peaks per platelet when platelets in a flow chamber come into contact with immobilized VWF. The first peak is dependent on the interaction of platelets with VWF and has been interpreted as being an important signal for activation of αIIbβ3 . To determine whether VWF is required in vivo for platelet activation independent of its role in platelet accumulation, platelet activation at the site of vascular injury was studied in VWF –/– mice. Platelet activation was normal, whether analyzed as calcium mobilization per platelet or as calcium mobilization per thrombus (Fig. 2B). These results indicate that in a thrombin-initiated thrombosis model, platelet activation is independent of VWF .
Intracellular signaling and calcium mobilization in platelets
Outside-in αIIbβ3 signaling is required for normal platelet thrombus formation and is triggered by c-Src activation via PTP-1B . Studies of PTP-1B-deficient mouse platelets in vitro indicate that PTP-1B is required for fibrinogen-dependent platelet spreading and clot retraction. In vivo thrombus formation is reduced in PTP-1B null mice, a manifestation of ineffective calcium mobilization during platelet activation. It would appear that PTP-1B is a regulator for the initiation of outside-in αIIbβ3 signaling.
Bile salt-dependent lipase (BSDL) has structural homology to the V3 loop of HIV-1 that binds to the chemokine receptor 4, CXCR4. Therefore, we hypothesized that BSDL might interact with CXCR4 present on platelets and influence platelet thrombus formation. In vitro BSDL induces calcium mobilization and enhances both platelet aggregation and spreading induced by thrombin . These effects are abolished when CXCR4 is inhibited. In vivo, endogenous mouse BSDL accumulates during thrombus formation. When CXCR4 is blocked, the accumulation of endogenous BSDL is inhibited and thrombus size is significantly reduced. In BSDL–/– mice calcium mobilization into platelets and thrombus formation are significantly attenuated, whereas fibrin generation is comparable with wild-type mice.
Gas 6, a γ-carboxyglutamic acid-containing membrane protein, is present on the platelet membrane and binds to a receptor tyrosine kinase, Axl. Gas 6 amplified platelet aggregation and secretion responses to platelet agonists . Deficiency of Gas 6, either in a Gas 6–/– mouse or using blocking antibodies to Gas 6, protected mice from fatal thrombosis but did not impair normal hemostasis. Gas 6 may participate in amplifying signaling events induced by agonists.
PECAM-1 is a cell adhesion molecule found on endothelial cells and platelets. PECAM-1 null mice formed larger arterial thrombi more rapidly than wild-type mice . Chimeric mice indicate that platelet PECAM-1 is the critical component, and suggest that PECAM-1 is involved in negative regulation of thrombus formation.
Inevitably, additional proteins will be identified that impact on signaling events during thrombus formation.
The platelet synapse
The platelet-platelet synapse, classically described as dominated by the integrin αIIbβ3 and its interaction with fibrinogen, appears to involve numerous adhesion molecules besides αIIbβ3. It is becoming increasingly clear that additional proteins contribute to platelet–platelet interaction, and these observations have emerged from in vivo experiments.
The Signaling Lymphocyte Activating Molecule (SLAM) adhesion receptors are expressed on platelets . SLAM-deficient platelets showed defective aggregation and SLAM null mice exhibit delayed thrombus formation in vivo. It has been proposed that SLAM plays a secondary role in the formation of the platelet–platelet synapse.
CD40L, a transmembrane platelet granule protein, is expressed on the plasma membrane of activated platelets. Mice deficient in CD40L show an in vivo defect in thrombus formation . These CD40L–/– mice exhibit delayed arterial occlusion and thrombus instability. CD40L may also be an αIIbβ3 ligand required for stable formation of arterial thrombi.
Platelets express Eph4 and EphB1 and at least one ligand, ephrinB1. During αIIbβ3-mediated platelet aggregation, Eph/ephrin interactions on platelet surfaces contribute to platelet–platelet interaction . These interactions favor thrombus growth and stability.
Platelets express semaphorin 4D and both of its receptors, molecules that promote thrombus formation . The surface expression of semaphorin 4D and CD72 increases during platelet activation, followed by the gradual shedding of the semaphorin 4D extracellular domain. Mice that lack semaphorin 4D have delayed arterial occlusion after vascular injury in vivo, and their platelets show impaired collagen responses in vitro.
The platelet synapse is likely to involve many proteins, and these protein–protein interactions occur early, intermediate and late in thrombus formation. They probably play an important role in platelet–platelet docking, platelet signaling and thrombus regulation.
Given the complexity of the hemostatic system, it has been necessary to move to animal models to explore the function of the proteins and cellular elements that are critical to thrombus formation. These studies will continue to allow revision of old concepts and identify new pathways and new components that have not been previously identified within this host defense mechanism.
Disclosure of Conflict of Interest
The authors state that they have no conflict of interest.