Denisa D. Wagner, Peter C. Burger
Abstract—For many years it has been known that platelets play an important role in thrombosis and hemostasis. In recent times, however, it has become evident that platelets also have relevant functions in inflammation. It was shown that thrombosis and inflammation share several key molecular mechanisms and in fact are 2 intrinsically linked processes. In this review, we intend to give a short overview with emphasis on work stemming from our laboratory. (Arterioscler Thromb Vasc Biol. 2003;23:2131-2137.)
Key Words: platelets䡲inflammation䡲thrombosis䡲atherosclerosis䡲hemostasis
Endothelial Response to Injury and
Platelet Adhesion
One linkage between thrombosis and inflammation can be seen at the molecular and cellular levels in the endothelium. The primary molecules responsible for initiation of platelet and leukocyte adhesion, von Willebrand factor (VWF) and P-selectin, respectively, are stored in the same storage gran-ules called Weibel-Palade bodies.1– 4 VWF is essential for
Weibel-Palade body formation,5 and therefore mice with
VWF deficiency cannot store P-selectin in endothelium, leading to its partial degradation. This makes the mice less responsive to inflammatory stimuli.6 On cellular activation
with either prothrombotic secretagogues, such as thrombin, or proinflammatory secretagogues, such as histamine, Weibel-Palade bodies fuse with the cell membrane and expose membrane-bound P-selectin and soluble VWF at the cell surface.7
Fairly recently it was found that both VWF and P-selectin, freshly secreted from activated endothelium, mediate platelet interaction with the endothelial surface. As to which of these 2 molecules will be the primary player seems to depend on the shear rate of the particular vessel. Andre´ et al8 showed
that in stimulated veins with low shear rates, large numbers of resting platelets translocate (a stop-and-go phenomenon) on VWF that is transiently bound to the endothelial surface. Under these conditions, platelet glycoprotein (GP) GPIb␣ mediates platelet adhesion. This transient adhesion process is likely terminated by the cleavage of VWF multimers by ADAMTS13,9the enzyme deficient in patients with inherited
thrombotic thrombocytopenic purpura.10 At higher venular
shear rates, activated and nonactivated platelets are able to roll on activated endothelial cells (similar to the well-known mechanism of leukocyte rolling), a process mediated by P- and E-selectin.11,12 P-selectin glycoprotein ligand-1
(PSGL-1), the main ligand of P-selectin, expressed on
leuko-cytes was also found on platelets and was shown to be at least partially responsible for this phenomenon.13GPIb␣is another
ligand of P-selectin and may therefore also support platelet rolling on activated endothelium.14P-selectin– dependent
in-teraction of platelets with the vessel wall was demonstrated during ischemia and reperfusion.15
Platelets in Thrombosis and Hemostasis
A severe vascular injury will lead to deendothelization and exposure of subendothelium. VWF, previously produced and released by the endothelium or adsorbed from plasma onto exposed tissue, is crucial for platelet adhesion, in particular in vessels with high shear rates, such as arteries and arte-rioles.16 –18 Here also VWF binds to its platelet membrane
receptor, GPIb-IX-V, establishing a transient bond that slows down platelets and thus facilitates their activation.19Absence
of VWF, as investigated in VWF-deficient mice by intravital microscopy, significantly delays platelet deposition on a ferric chloride–injured vessel wall and significantly increases the time required for the formation of a first thrombus.20,21In
humans, absence of the GPIb-IX-V complex results in a severe bleeding disorder known as Bernard Soulier syn-drome, which, besides impaired VWF binding, is character-ized by thrombocytopenia and giant platelets.22
After tethering, rapid conversion to stable platelet adhesion is required to promote thrombus formation. This process is primarily mediated by the interaction of platelet surface– expressed integrins such as integrin ␣21 and the
immunoglobulin-like receptor GPVI with collagen. These interactions promote platelet activation and as a consequence promote a shift to a high-affinity state of the major platelet integrin␣IIb3, which mediates platelet aggregation. Collagen
binding also induces the release of adenosine diphosphate and thromboxane A2, platelet agonists that additionally activate
adherent platelets in an autocrine manner by amplifying
Received July 14, 2003; revision accepted August 28, 2003.
From The CBR Institute for Biomedical Research and Department of Pathology, Harvard Medical School, Boston, Mass. This in an invited article based on the Plenary Lecture given at the 4th Annual ATVB Conference, May 8, 2003, Washington D.C.
Correspondence to Denisa D. Wagner, The Center for Blood Research, 800 Huntington Ave, Boston, MA 02115. E-mail [email protected]
(Arterioscler Thromb Vasc Biol. 2003;23:2131-2137.)
© 2003 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org DOI: 10.1161/01.ATV.0000095974.95122.EC
integrin adhesiveness and thereby promote thrombus growth.23–25 The serine protease thrombin generated by the
coagulation cascade is another important platelet agonist. Besides activating platelets through cleavage of its G-pro-tein–linked protease-activated receptors,26thrombin can also
cleave GPV of the GPIb-IX-V complex, which allows throm-bin throm-binding to GPIb␣, a process that also results in platelet activation.27 Absence of GPV on platelets, as tested in
GPV-null mice, results in enhanced platelet responsiveness to thrombin and in faster thrombus growth in vivo.28
The absence of ␣IIb3 integrin in human causes a severe
bleeding disorder called Glanzmann thrombasthenia.29
Ob-servation of injured arterioles of 3-deficient mice showed
defects in stable platelet adhesion and, even more impor-tantly, complete absence of aggregate formation (Hynes and Wagner, unpublished observations, 2002). Thus,3integrin
is absolutely crucial for platelet crosslinking. Fibrinogen was generally considered the main ligand crosslinking the␣IIb3
integrins on neighboring platelets.30 Surprisingly, in the
absence of fibrinogen, as tested in fibrinogen-deficient mice (Fg⫺/⫺), thrombi still form readily. Thrombi of these mice,
however, are unstable and fail to resist shear stress, which results in frequent embolization of the entire thrombus, with vessel occlusion downstream of the injured area.21 Clearly,
fibrinogen/fibrin is required to secure thrombus stability. In addition to the important function of the fibrin meshwork, which ensures thrombus anchoring at the site of injury, we recently found that thrombi are stabilized by the interaction of the C-terminal␥chain of fibrinogen/fibrin with platelet␣IIb3
integrin, an interaction that has been demonstrated in vitro.31
Like Fg⫺/⫺mice, mice deficient in the last 5 amino acids of
the fibrinogen ␥ chain (Fg␥⌬5 mice)32 showed enhanced
embolization on vascular injury.33However, in contrast to the
Fg⫺/⫺ mice, the Fg␥⌬5 mice were able to form occlusive
thrombi at the site of injury because fibrin can still form in these animals.
The very rapid thrombus growth in the absence of fibrin-ogen questions the key role of fibrinfibrin-ogen in the formation of platelet-platelet aggregates in vivo. The ligand responsible for the fast growing thrombi in the fibrinogen-deficient mice was not VWF. Mice deficient in both VWF and fibrinogen were still capable of forming thrombi. Platelets of fibrinogen-deficient mice21 as well as of Fg␥⌬5 mice33 accumulate
excessive amounts of fibronectin, indicating that fibronectin competes with fibrinogen for binding to ␣IIb3 , which
mediates internalization of these molecules into platelet
␣-granules. Fibronectin is known to support platelet adhesion and spreading.34However, the role of fibronectin in thrombus
formation and stabilization was not well defined. We there-fore tested in a conditional fibronectin knockout mouse model35whether fibronectin is an important ligand in
throm-bus growth. Deficiency of plasma fibronectin did not affect the initial platelet adhesion, but it resulted in a delay of several minutes in thrombus formation. Thrombi of fibronectin-deficient mice continuously shed platelets or small platelet clumps, leading to significantly slower throm-bus growth than seen in wild-type mice. Therefore, fibronec-tin is an important ligand mediafibronec-ting platelet-platelet interac-tions within growing thrombi.36
Thus, from the work with transgenic mice and previous in vitro work under flow, a clearer model of thrombus growth in high shear rate conditions emerges. VWF mediates the early platelet adhesion to the injured site. Fibronectin rapidly cross-links platelets by binding to activated␣IIb3 integrins.
Fibrin is generated and anchors the growing thrombus to the site of injury. Platelets attach to the fibrin/fibrinogen mesh-work also by ␣IIb3 integrin, which stabilizes the thrombus
and slows down its growth. Finally, at very high shear rates, near vessel occlusion, VWF is required again because throm-bus growth in arterioles of VWF-deficient mice tends to arrest before vessel occlusion, with high-shear channels remaining open.21Although this has not been demonstrated in
vivo, VWF likely fortifies the thrombi by binding to GPIb␣, in addition to the␣IIb3-mediated linkages.37
In recent work in collaboration with David Phillips, we were able to identify another important new ligand for␣IIb3,
CD40L.38This molecule represents yet another link between
inflammation and thrombosis. CD40L, a transmembrane protein of the tumor necrosis factor (TNF) family, is ex-pressed not only on cells of the immune system39but also on
activated platelets.40 It is stored in platelets and gets
exter-nalized and shed on platelet activation. CD40L-deficient mice exhibited delayed vessel occlusion and frequent emboliza-tion, a phenotype that could be rescued by the injection of recombinant soluble CD40L. CD40L, by binding to the 3
integrin through its KGD amino acid sequence, may directly activate the ligand-binding activity of ␣IIb3, as reported for
RGD peptides.41Because of its small size, it is less likely that
CD40L directly cross-links platelets like other␣IIb3ligands.
Because fibrin formation by thrombin is very important for thrombus stability, we will discuss a newly understood role of P-selectin, the adhesion receptor for leukocytes, in this process. Two observations first pointed to a role of P-selectin in coagulation. First, Palabrica et al42showed that leukocytes
are recruited into growing thrombi and promote fibrin depo-sition that is inhibited by antibodies to P-selectin. Second, Hartwell et al43generated a knock-in mouse of P-selectin that
lacks the cytoplasmic domain (⌬CT mouse) and is character-ized by high levels of soluble P-selectin in plasma. Blood of these mice clotted (formed fibrin) faster, indicating that the shedding of P-selectin generated a procoagulant state in the animals. Furthermore, excessive fibrin deposition on platelet thrombi in ex vivo flow chamber studies was observed. In vivo, the hemorrhage produced by a local Schwarzmann reaction was significantly smaller in⌬CT than in wild-type mice because of a protective layer of fibrin deposited rapidly on the inside of the vessel wall. This effect could be reproduced by infusion of a chimeric P-selectin immunoglob-ulin fusion protein (P-sel-Ig) into wild-type mice, as shown in Figure 1.
Celi et al44 showed that P-selectin, either expressed on
activated cells or purified, upregulates tissue factor (TF) expression on monocytes in vitro, and more recently TF was found to circulate in blood as part of microparticles.45We
showed that P-sel-Ig induced leukocytes to form such TF-containing microparticles and that these microparticles were responsible for the procoagulant activity in the⌬CT mice.46
Higher than normal levels of soluble P-selectin in blood were found in thrombotic consumptive platelet disorders, such as disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, or heparin-induced thrombocyto-penia, disorders that are all associated with thrombotic and thromboembolic complications.47Furthermore, higher levels
of soluble P-selectin are predictive of future cardiovascular events.48,49These findings support the concept of P-selectin
having procoagulant activity.
Hrachovinova et al50 recently found that P-sel-Ig can
induce the formation of TF containing microparticles in human as well as in mouse blood and that this is mediated through binding to PSGL-1 on leukocytes. Compared with wild-type mice, mice that were deficient in PSGL-1 (PSGL-1⫺/⫺mice) produced fewer microparticles both spontaneously
in old age as well as after P-sel-Ig infusion. Thus, by inducing the generation of soluble TF, P-selectin and its receptor PSGL-1 play an important role in hemostasis.50
Because TF-containing microparticles were generated from leukocytes, their adhesion interactions are similar to those of the cell of origin. It was shown by intravital microscopy that native microparticles might be directly in-volved in thrombin generation because they specifically bind to growing thrombi at sites of vascular injury.50In vitro, this
was shown to be mediated by P-selectin and a carbohydrate ligand with some involvement of TF.51 When infused into
mice, in vitro–produced microparticles from monocytes were also found to be recruited to thrombi in a P-selectin/PSGL-1– dependent manner.52
These observations have therapeutic implications. Inhibi-tion of P-selectin/PSGL-1 interacInhibi-tion would reduce proco-agulant activity. This is likely why recombinant soluble PSGL-1 promotes thrombus dissolution4,53,54 and why
P-selectin– deficient mice have a slightly prolonged bleeding time.55 On the other hand, increasing P-selectin/PSGL-1
interactions would have an opposite effect. This too may be desirable in certain clinical situations. In a mouse model of hemophilia A,56 infusion of P-sel-Ig produced a 20-fold
increase in TF-containing microparticles, which significantly facilitated fibrin formation and normalized the impaired tail-bleeding time of these mice within 6 hours.50 P-sel-Ig
could become a new method to treat hemophilia patients, in particular those producing alloantibodies to factor VIII or IX.
Platelets in Inflammation
Inflammation leads to an imbalance between procoagulant and anticoagulant properties of the endothelium that can lead to a local stimulation of the coagulation cascade. TNF-␣, the first proinflammatory cytokine released at the site of infec-tion, is a potent inducer of the immune defense mechanism and a mediator of leukocyte recruitment.57 It promotes a
procoagulant state by inhibiting synthesis of the anticoagulant protein C58and by eliciting TF production on the endothelium
and monocytes, thereby stimulating thrombin and fibrin formation.59 TNF-␣ has pleiotropic effects, like most
cyto-kines. Nevertheless, it was rather surprising that TNF-␣, at concentrations found in lethal or sublethal sepsis, strongly inhibited thrombus growth in the ferric chloride vascular injury model and prolonged bleeding time of treated mice (B. Cambien, unpublished data, 2003). This transient (lasting less than 2 hours) powerful antithrombotic effect of TNF-␣is not mediated by a platelet TNF receptor but rather through the stimulation of inducible NO synthase by TNF receptors in the vessel wall with consecutive release of NO, a strong inhibitor of platelet activation.60Thus, perhaps in the early phases of
infection, when leukocyte recruitment is of primary impor-tance to prevent bacterial spread, the formation of thrombi obstructing blood flow is not desired. In addition, NO produced constitutively by endothelial cells significantly inhibits Weibel-Palade body release, thus reducing platelet adhesion to endothelium.61
Inflammation is also characterized by a multitude of interactions between leukocytes, endothelial cells, and plate-lets. Irrespective of its etiology, inflammation causes endo-thelial activation. Activated endoendo-thelial cells express cell adhesion molecules such as P- and E-selectin, which mediate leukocyte rolling, the first step in the cell-adhesion cas-cade.62,63 Rolling leukocytes become activated by
chemo-kines such as monocyte chemoattractant protein 1 and RAN-TES, presented by the activated endothelial surface. The activated leukocytes can bind to other endothelial adhesion molecules, such as intercellular adhesion molecule-1 and vascular adhesion molecule-1, and start transmigrating into the vascular intima. Targeted disruption of the P- and E-selectin gene in the mouse results in marked inhibition of leukocyte rolling and delayed recruitment of monocytes into sites of inflammation and in enhanced susceptibility to infection.64 – 67
Figure 1.Effect of sP-sel-Ig on fibrin distribu-tion at the Shwartzman reacdistribu-tion hemorrhagic lesion site. A, Micrographs showing fibrin depo-sition (F) in paraffin sections from the Shwartz-man lesion sites. Tissue sections from wild-type mice treated with IgG1 (Ig-control) or P-sel-Ig were immunostained with antibodies to fibrino-gen. Note the presence of hemorrhage (H) in the section from the IgG1-treated animals and a diffuse staining for fibrin inside and outside the vessel. A strong fibrin staining (F) is found on the luminal face of the vessel wall in the P-sel-Ig–treated mice without detectable fibrin deposition in the surrounding tissue. White arrowheads point to the vessel wall. Reprinted with permission from Reference 46.
Atherosclerosis is a typical example of a chronic inflam-matory process.68Absence of P-selectin was shown to reduce
and delay atherosclerotic lesion formation in atherosclerosis-prone LDLR-deficient69 or apoE-deficient70,71 mice. More
recently, in a bone marrow transplant animal experiment, we demonstrated that not only endothelial P-selectin but also platelet P-selectin contributes to atherosclerotic lesion forma-tion in the apoE-deficient mice, demonstrating direct platelet and platelet P-selectin involvement in the atherosclerotic process.72Interestingly, P-selectin also influences lesion
mat-uration in a dose-dependent manner (Figure 2). P-selectin mediates rosetting of monocytes and neutrophils with circu-lating activated platelets,73 an interaction that increases
monocyte adhesion to endothelial cells and may finally facilitate macrophage accumulation in the vessel wall.74
Platelets adherent to subendothelial matrix also support leu-kocyte rolling, adhesion, and transmigration through the interaction of their P-selectin with leukocyte PSGL-1.75–77
The process of leukocyte rolling allows platelet activating factor to stimulate leukocyte integrin Mac-1, become acti-vated, and mediate firm leukocyte adhesion through binding to fibrinogen that is also bound to the platelet␣IIb3.78During
adhesion to endothelium, platelets are activated and release
proinflammatory cytokines, such as CD40L40 and
interleukin-I,79that can further stimulate the endothelium.
Adhesion of platelets or platelet-derived microparticles80 is
followed by activation of endothelial nuclear factor-B and nuclear factor-B–regulated genes, such as monocyte che-moattractant protein-1, ␣v3, intercellular adhesion
molecule-1, and vascular cellular adhesion molecule-1, or genes that play important roles in chemotaxis and transmi-gration of monocytes.81,82
Activated platelets also express and secrete the chemokines CCL5 (RANTES) and CXCL4 (platelet factor 4), which are deposited in a P-selectin– dependent manner on microvascu-lature, aortic endothelium, and monocytes.83 Deposition of
these proinflammatory cytokines results in activation of
monocyte integrins and in increased monocyte recruitment to atherosclerotic lesions.84
Massberg et al85 showed in vivo that platelet adhere to
carotid endothelium at an early stage of atherosclerosis in hypercholesterolemic apoE-deficient mice. Prolonged block-ade of platelet adhesion by antibodies against GPIb␣ signif-icantly attenuated atherosclerotic lesion formation in these animals, indicating that platelet GPIb␣ interaction with its ligands VWF and P-selectin, which are found on activated endothelial cells,8,14might be responsible for platelet binding.
This indicates that VWF could play not only an important role in thrombosis and hemostasis but could also be involved in atherosclerotic lesion development. Indeed, LDLR- or apoE-deficient mice that were also VWF-deficient showed a transient delay in atherosclerotic lesion development, with fewer monocytes in their lesions than wild-type control animals of the same background.86Interestingly, absence of
VWF affected mainly lesion distribution. VWF deficiency protected the animals from lesion development in areas with disturbed flow, such as arterial branch points of renal and mesenteric arteries and the aortic sinus. VWF may participate in the recruitment of monocytes directly through the VWF propolypeptide87or indirectly by recruiting platelets that may
facilitate leukocyte adhesion.8There is also a possibility that
VWF, through interaction with 3 integrin, is part of the
mechanotransduction pathway mediating the shear stress responses in endothelium. Thus, the exact mechanism respon-sible for the bizarre lesion distribution in VWF-deficient mice remains to be established.
Platelets, once recruited to atherosclerotic lesions, contain a variety of molecules that can additionally promote chemoat-traction of leukocytes (platelet activating factor, macrophage inflammatory protein (MIP)-1␣, and cationic proteins), stim-ulate smooth muscle cell and fibroblast proliferation (TGF-, platelet-derived growth factor, and serotonin), and promote collagen synthesis. Platelets with their cytokines thereby contribute directly to lesion progression and maturation.88As
Figure 2.Immunohistochemical staining for␣-actin shows positive SMCs in ath-erosclerotic lesions. Lesions of apoE-deficient mice that expressed P-selectin on platelets and endothelium (wt-wt) (A), endothelium only (ko-wt) (B), and neither platelets nor endothelium (ko-ko) (C); bar⫽0.2 mm. D,␣-actin–positive SMCs within lesions were counted on 5 aortic sinus sections 80m apart. Dots repre-sent the mean number of SMCs per sec-tion for each animal. Black dots (䢇) cor-respond to animals that lack endothelial P-selectin (ie, wt-ko, ko-ko). Bars show mean⫾SEM to each group. Atheroscle-rotic lesions of the chimeric animals, expressing P-selectin only either on platelets or on endothelial cells, appear to have an intermediate content of SMCs. Reproduced with permission from Reference 72.
already mentioned above, activated platelets also shed large amounts of soluble CD40L.89CD40L/CD40 interaction plays
an important role in atherosclerosis because antibody to CD40L-inhibited lesion development in LDLR-deficient mice90 and CD40L-deficient mice on apoE-deficient
back-ground was extensively protected from atherosclerosis.91
With all of these activities of activated platelets, it is not surprising that repeated infusions of activated platelets pro-moted atherosclerosis in apoE⫺/⫺mice. Here, too, the effect
was dependent on the presence of platelet P-selectin.84Thus,
3 independent studies using very different experimental approaches and published almost simultaneously demon-strated that platelets and their adhesion receptors play an important role in atherosclerotic lesion formation.72,84,85
Conclusions
Thrombosis and inflammation are intricately linked (Figure 3). The examples discussed above show that some of the same cellular and molecular players participate in both processes. On vascular injury, platelets cover the exposed subendothelial matrix and mediate additional platelet and leukocyte recruitment. To stop bleeding, they provide the required surface for the binding of leukocyte-derived micro-particles containing tissue factor for a localized induction of the coagulation cascade. Platelets also release microparticles that mediate leukocyte-leukocyte and leukocyte– endothelial cell interactions. Most of these mechanisms play a role in inflammation as well. Activated platelets increase leukocyte adhesion to the endothelium and promote leukocyte activa-tion through deposiactiva-tion of chemokines on the endothelium. This enables leukocytes to firmly attach to the vessel wall and finally to transmigrate into the subendothelial tissue (Figure
3). The role of platelets in chronic inflammatory diseases such as atherosclerosis has now been convincingly demon-strated. Thus, platelets are central to both thrombosis and inflammation.
Acknowledgments
The National Institutes of Health grants PO1 Hl56949, R01 Hl53756, and R37 HL41002 (to D.D.W.) supported the work of the authors’ laboratory. P.C.B. was supported by a stipend from the Swiss National Science Foundation. The authors thank Wolfgang Berg-meier for critical reading of the manuscript and Janet Diver and Tomoko Tagaki for assistance in preparing the manuscript.
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