Journal of Thrombosis and Haemostasis, 13 (Suppl. 1): S40–S46
DOI: 10.1111/jth.12976
INVITED REVIEW
Platelets and their microparticles as key players in pathophysiological responses D . V A R O N and E . S H A I Coagulation Unit, Department of Hematology, Hadassah Medical Center, Jerusalem, Israel
To cite this article: Varon D, Shai E. Platelets and their microparticles as key players in pathophysiological responses. J Thromb Haemost 2015; 13 (Suppl. 1): S40–S6.
Summary. Platelets are known to play a central role in primary hemostasis as well as in the pathophysiology of thrombotic disorders. However, in addition to hemostasis, platelets are involved in a variety of pathophysiological responses including immune responses, inflammation, angiogenesis, tissue regeneration, and cancer metastasis. Recent studies revealed a significant role for plateletderived microparticles (PMP), in these responses. PMP communicate with, and deliver signals to, other cells, induce signals, and change their phenotype during inflammation, angiogenesis, and tumor metastasis. The current report describes the recent development in this field with a focus on the role of platelets and PMP in all of the above responses. Keywords: angiogenesis stimulators; cancer; inflammation; cell-derived microparticles; platelets.
Introduction Platelets are known to play a central role in primary hemostasis as well as in the pathophysiology of thrombotic disorders. However, in addition to hemostasis, platelets were shown to be involved in a variety of pathophysiological responses including immune responses, inflammation, angiogenesis, tissue regeneration, and cancer metastasis [1]. Platelet a-granules contain different cytokines and growth factors, including angiogenesis-regulated agents such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and metalloproteinases (MMPs) [2,3]. On the other hand, platelets also contain angiostatin and endostatin – both potent inhibitors of angiogenesis, thrombospondin-1 (TSP1) an inhibitor of endothelial cell Correspondence: David Varon, Coagulation Unit, Department of Hematology, Hadassah Medical Center, Jerusalem 91120, Israel. Tel.: +972 2 677 7672; fax: +972 2 644 9580. E-mail: [email protected]
proliferation and capillary tube formation, and tissue inhibitors of metalloproteinases. These biologically active molecules that may be delivered to the endothelium and tissues upon platelet adhesion to the injured site of the vasculature make platelets effective promoters of cell growth and adhesion and blood vessel development. Microparticles (MP) are small plasma membrane vesicles (0.05–1 lm) shed from different cells upon activation or apoptosis [4]. The mechanism of MP formation is disruption of the machinery supporting asymmetry between the two phospholipid layers of cell membrane. Plateletderived microparticles (PMP) constitute the majority of the pool of MP circulating in the blood [5]. PMP contain a unique subset of proteins derived from the parent cell, and in recent years, it has become clear that PMP have important biological functions. The best characterized of these functions is their participation in blood coagulation by providing a source of tissue factor (TF) as well as negatively charged surfaces where clotting factor complexes can assemble [6]. However, other studies have suggested that PMP, isolated either from plasma or from supernatants of stimulated platelets, promote the expression of adhesion molecules on a variety of cells, stimulate the release of cytokines, alter vascular reactivity, induce inflammation and angiogenesis, and are involved in cancer metastasis [7,8]. It has been shown that PMP can express and transfer functional receptors from platelet membranes, such as glycoprotein IIb-IIIa (GPIIb-IIIa) and P-selectin, to different cell types [9] and, by that, enhance engraftment of hematopoietic stem/progenitor cells [10], as well as confer adhesive properties with circulating tumor cells (O. Dashevsky, E. Shai and D. Varon, unpublished data). In addition, PMP promote monocyte and neutrophil adhesion to the endothelium and stimulate COX-2 expression in monocytes and endothelial cells [11,12]. Furthermore, it was established that PMP could not only passively carry various proteins and receptors, but also chemoattract hematopoietic cells and stimulate their adhesion, survival, and proliferation [8]. Moreover, PMP © 2015 International Society on Thrombosis and Haemostasis
Platelets and their microparticles S41
activate intracellular signaling pathways such as ERK, PI3-kinase/Akt, and STAT proteins. These effects could only partially be overturned by heat and trypsin, suggesting that, in addition to intraparticle agents of protein nature, the lipid component of PMP could also be involved. The current report describes the role of platelets and PMP in immunity and inflammation, angiogenesis, tissue regeneration, and cancer progression. Immunity and inflammation Platelets have a broad repertoire of receptor molecules that enable them to recognize pathogen-associated molecular patterns and sense infection-induced inflammation. Different Toll-like receptors have been found on platelets. These receptors bind ligands from infectious pathogens and mediate microbial sensing and host defense mechanisms [13,14]. Platelets, along with their microparticles and with other inflammatory cells, can also facilitate microbial adhesion to endothelial cells (EC) in a variety of inflammatory diseases [15]. We have shown that in vitro treatment of EC with platelets significantly increases adherence of S. Aureus to the EC, both under static and moderate flow rate conditions [16]. These observations suggest a role for platelets in bacterial infection and colonization in the setting of endocarditis and other infections. More recent studies show the involvement of platelet factor 4 in bacteria-induced activation of platelets, through a common pathway that involves FccRIIA receptor that is present on the platelet surface. This interaction results in an inside-out signaling leading to integrin GPIIb-IIIa engagement and dense granule secretion [17]. Hu et al. [18] described the mechanisms of platelet interaction with monocytes, neutrophils, and lymphocytes, under static and flow conditions, suggesting different mechanisms for each of these interactions. Recently, it was demonstrated that recruited neutrophils scan for activated platelets through the selectin ligand P-selectin glycoligand-1 to initiate inflammation [19]. Although platelets exert antimicrobial effector mechanisms and initiate cross talk with the innate and adaptive immune factors, platelets may also induce injurious systemic responses in various infectious syndromes. Infection with dengue virus, for instance, may induce thrombocytopenia through two different mechanisms – impaired thrombopoiesis and peripheral platelet destruction via direct interaction [20]. Malaria is also associated with thrombocytopenia due to peripheral destruction, compromised platelet function and excessive sequestration of platelets in the spleen. During cerebral malaria, platelets were shown to be deleterious to the brain endothelium, by allowing binding between infected red blood cells and the endothelial cells [21]. However, other studies indicate a protective function for platelets in the early stages of erythrocytic infection, © 2015 International Society on Thrombosis and Haemostasis
by killing the parasite within the red cell. Deficient platelets and aspirin treatment reversed this effect [22]. Recently, it was demonstrated that activated platelets generate significant amounts of oxidized phospholipids, indicating that phospholipid oxidation is a regulated process of likely importance during physiological hemostasis and in acute response to injury. [23]. Oxidized low-density lipoproteins (ox-LDL) have an important role in the development of inflammatory-related vascular disease, such as coronary artery disease and atherosclerosis. We investigated the effects of homocysteine and ox-LDL on EC thrombogenicity. Treatment of EC with either homocysteine (1 or 10 mmol L1 for 16 h) or ox-LDL (100 lg mL1 for 16 h) resulted in a 2- to 3-fold enhancement in platelet adhesion under flow conditions at a shear rate of 350 s1. The enhancement in platelet adhesion induced by homocysteine was absolutely dependent on fibrin formation. Homocysteine treatment has significantly increased the cell surface TF activity, while oxLDL treatment upregulated intercellular adhesion molecule (ICAM)-1 expression and had no significant effect on endothelial TF activity. The homocysteine-induced enhancement in platelet adhesion was significantly reduced by blockade of the EC TF activity as well as by inhibition of the EC avb3 integrin. On the other hand, oxLDL-induced enhancement in platelet-EC adhesion was greatly inhibited by blocking ICAM-1 or avb3, but remained unaffected by inhibition of TF activity. Preincubation of platelets with the GPIIb-IIIa antagonist ReoPro virtually abolished the enhancing effect of both homocysteine and ox-LDL. These results suggest that homocysteine and ox-LDL might increase EC thrombogenicity by distinct mechanisms: homocysteine, by inducing TF activity; and oxLDL, by upregulating ICAM-1, both of which enhance GPIIb-IIIa/fibrinogen-dependent platelet adhesion to EC. The avb3 integrin, although not affected by EC stimulation, seems to play a crucial role in platelet-EC interaction regardless of the mechanism of EC perturbation [24]. Activated platelets have many mechanisms for delivering signals to target cells involved in inflammatory interactions. These diverse mechanisms include platelet secretion of mediators that affect local innate immune response both locally and through the secretion of PMP [25]. Platelets can also modulate adaptive immunity through their microparticles, at sites distant from the location of platelet activation [26]. Boilard [27] explored the mechanisms by which platelets are activated to release microparticles in the context of arthritis. Using genetically deficient mice and pharmacologic blockade, they showed that platelets form MP and participate in arthritis pathophysiology via stimulation of the collagen receptor GPVI. They further showed that other pathways such as thromboxane A2 stimulation, ligation of the P2Y12, or GPIb-IX receptor do not regulate platelet MP generation in inflammatory arthritis. MP
S42 D. Varon and E. Shai
formation from platelets can be induced by high sheer stress alone. However, interaction of blood cells with TNF-a-activated EC under flow conditions can lead to changes in the proportion of circulating MP [28]. More recently, it was shown that some PMP contain respiratory-competent mitochondria. These plateletsecreted mitochondria serve a substrate of phospholipase A2 IIA (that can also be secreted by platelets) to yield inflammatory mediators that promote leukocyte activation [29]. As suggested before, this study also shows further evidence that PMP are heterogeneous in their biological activities depending on the cargo they carry. Angiogenesis and tissue regeneration The role of PMP in blood vessel development was first shown by Kim [7]. They reported that PMP promote proliferation of EC. This effect was mediated by a concerted action of VEGF, FGF-2, and a lipid component of PMP, most likely sphingosine-1-phosphate. This lipid constituent seems to mediate also the anti-apoptotic and chemotactic effects of PMP on EC as well as PMP-induced tubule formation. We showed that platelets stimulate formation of blood vessels in vitro in the rat aortic ring model via VEGF and bFGF. Blockage of platelet factor-4 promoted this effect. Platelet releasate was found to induce EC chemotaxis, mediated by a concerted action of intraplatelet bFGF, PDGF, VEGF, and heparanase. A concomitant effect of bFGF and VEGF seemed to be essential for the entire process of vessel formation, while PDGF and heparanase were effective only at the migration stage. We concluded that platelets affect different stages of the angiogenic response with a trend to a pro-angiogenic net effect despite the presence of angiogenesis inhibitors [30]. The pro-angiogenic effect of platelets and PMP raises the question of what is the mechanism for this effect in view of the presence of angiogenic inhibitors (along with activators) in platelets a-granules. One potential explanation was offered by Italiano [31], who showed differential localization of angiogenic cytokines in different granules. This idea was further established by a different group who showed by electron tomography and 3D analysis that the a-granule population is morphologically heterogeneous. They visualized long, highly curved membrane strands, connected to spherical domains and confirmed that these tubules represent a-granules. They proposed that cargo is differentially released from tubular granules compared with spherical granules [32]. Jonnalagadda et al. showed that platelet secretion was kinetically heterogeneous in response to four different agonists. They found that the extent and rate of release are related to agonist potency with thrombin inducing the most rapid release of the highest number of cargo. However, cargo with opposing actions had similar release profiles, suggesting that platelet release can be calcified to
at least 3 classes of release processes differing in rate, but the distribution of cargo into each class is random [33]. It has been shown that PMP also differ in their content of exposed membrane anionic phospholipids and their intrinsic procoagulant potential, depending on the stimulant used to generate them [34]. We investigated the proteome of PMP produced by either thrombin stimulation or by shearing the platelets under arterial flow conditions (1800 s1). These studies revealed significant changes in the expression of several proteins related to cytoskeleton and platelet shape change machinery [35]. In a recent proteome analysis, Ve0 lez et al. [36] showed that the platelet secretome varies with the stimulus by comparing the platelet releasate following platelet activation with thrombin or collagen. These results are line with the studies that show that the release of angiogenesis regulatory proteins is modulated by physiological processes [31,37]. These studies demonstrated that activation of platelets with ADP stimulated the release of VEGF, but not endostatin, whereas thromboxane A2 released endostatin but not VEGF. They also showed that platelet releasates, generated by activation with ADP, promoted migration and formation EC tubules in in vitro angiogenesis models. We have demonstrated that PMP induce sprouting both in vitro and in vivo (Fig. 1) to a degree comparable with that of whole platelets [38]. Moreover, application of PMP may have a therapeutic potential. Intramyocardial injection of PMP markedly elevated the amount of novel capillaries developed in the heart muscle in the background of ischemia (LAD ligation in a rat model, Fig. 2). Thus, PMP may further support the platelet-induced angiogenic response operating relatively independently of platelets. Alternatively, PMP may be a tool or a mediator through which platelets exert their pro-angiogenic effect given that various released molecules traditionally considered as soluble are in fact bound to MP [39]. The influence of PMP on the angiogenic activity of endothelial progenitor cells (EPCs) was shown by Prokopi [40]. Endothelial tube formation was stimulated by conditioned medium of EPC cultures; this effect was attenuated by removing MPs from the conditioned medium by filtration, ultracentrifugation, or inhibition of the formation of the platelet GPIIb-IIIa integrin complex. This study also emphasized the misinterpretations that might arise from PMP-mediated exchange of antigens. They showed that mononuclear blood cells uptake platelet-derived microparticles and exchange marker proteins, replicating the phenotype of EPCs. This might have important implications for determination of progenitor cells for clinical trials in stem cell therapy. We further studied the potential effect of PMP in a stroke model. We hypothesized that tissue regeneration secondary to activation of endogenous neural stem cells (eNSC) can be enhanced by delivering platelets and PMP to the ischemic brain. To examine these potential therapeutic effects, we injected platelet poor plasma © 2015 International Society on Thrombosis and Haemostasis
Platelets and their microparticles S43
A
1
2
3
4
6
8
#
# ^
6
*
4
Infarction+PMP
2 0
0
20
30
50
PMP, µg mL–1 protein
100
VEGF + bFGF
Fig. 1. Platelet-derived microparticles induce angiogenesis in the aortic ring model. A. PMP in the indicated concentrations were added to the rings. After 7 days of incubation, the preparations were fixed, stained, and photographed. The following samples are shown: (1) negative control, (2) PMP 20 mg mL1, (3) PMP 30 mg mL1, (4) PMP 50 mg mL1, (5) PMP 100 mg mL1 and (6) VEGF 50 ng mL1 + bFGF 50 ng mL1. Representative experiment out of 7; bar 0.5 mm. B. Quantitative analysis of the pro-angiogenic effect of PMP. The experimental series were compared using unpaired Student’s t-test. *P < 0.02; vs. negative control (PMP 0), #P < 0.002 vs. negative control; ^P < 0.001 vs. negative control [38].
(PPP), FGF-2, PMP, and platelet lysate to the lateral ventricles after permanent middle cerebral artery occlusion (PMCAO) in rats. The animals were tested with the neurological severity score, and infarct volumes were measured at 90 days post-PMCAO. Immunohistochemistry was used to determine the fate of newborn cells and to count blood vessels in the ischemic brain. Platelets and PMP significantly increased eNSC proliferation and angiogenesis in the subventricular zone (SVZ) and in the perilesion cortex. Functional outcome was significantly improved, and injury size was significantly reduced in rats treated with platelet lysate suggesting additional neuroprotective effects (Fig. 3). Thus, a local delivery of platelet lysate or PMP to the lateral ventricles induces angiogenesis, neurogenesis, and neuroprotection and reduces behavioral deficits after brain ischemia [41,42]. Cancer progression The first report on the potential role of platelets in cancer progression descried the correlation between lung © 2015 International Society on Thrombosis and Haemostasis
200 160
P
DOI: 10.1111/jth.12976
INVITED REVIEW
Platelets and their microparticles as key players in pathophysiological responses D . V A R O N and E . S H A I Coagulation Unit, Department of Hematology, Hadassah Medical Center, Jerusalem, Israel
To cite this article: Varon D, Shai E. Platelets and their microparticles as key players in pathophysiological responses. J Thromb Haemost 2015; 13 (Suppl. 1): S40–S6.
Summary. Platelets are known to play a central role in primary hemostasis as well as in the pathophysiology of thrombotic disorders. However, in addition to hemostasis, platelets are involved in a variety of pathophysiological responses including immune responses, inflammation, angiogenesis, tissue regeneration, and cancer metastasis. Recent studies revealed a significant role for plateletderived microparticles (PMP), in these responses. PMP communicate with, and deliver signals to, other cells, induce signals, and change their phenotype during inflammation, angiogenesis, and tumor metastasis. The current report describes the recent development in this field with a focus on the role of platelets and PMP in all of the above responses. Keywords: angiogenesis stimulators; cancer; inflammation; cell-derived microparticles; platelets.
Introduction Platelets are known to play a central role in primary hemostasis as well as in the pathophysiology of thrombotic disorders. However, in addition to hemostasis, platelets were shown to be involved in a variety of pathophysiological responses including immune responses, inflammation, angiogenesis, tissue regeneration, and cancer metastasis [1]. Platelet a-granules contain different cytokines and growth factors, including angiogenesis-regulated agents such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and metalloproteinases (MMPs) [2,3]. On the other hand, platelets also contain angiostatin and endostatin – both potent inhibitors of angiogenesis, thrombospondin-1 (TSP1) an inhibitor of endothelial cell Correspondence: David Varon, Coagulation Unit, Department of Hematology, Hadassah Medical Center, Jerusalem 91120, Israel. Tel.: +972 2 677 7672; fax: +972 2 644 9580. E-mail: [email protected]
proliferation and capillary tube formation, and tissue inhibitors of metalloproteinases. These biologically active molecules that may be delivered to the endothelium and tissues upon platelet adhesion to the injured site of the vasculature make platelets effective promoters of cell growth and adhesion and blood vessel development. Microparticles (MP) are small plasma membrane vesicles (0.05–1 lm) shed from different cells upon activation or apoptosis [4]. The mechanism of MP formation is disruption of the machinery supporting asymmetry between the two phospholipid layers of cell membrane. Plateletderived microparticles (PMP) constitute the majority of the pool of MP circulating in the blood [5]. PMP contain a unique subset of proteins derived from the parent cell, and in recent years, it has become clear that PMP have important biological functions. The best characterized of these functions is their participation in blood coagulation by providing a source of tissue factor (TF) as well as negatively charged surfaces where clotting factor complexes can assemble [6]. However, other studies have suggested that PMP, isolated either from plasma or from supernatants of stimulated platelets, promote the expression of adhesion molecules on a variety of cells, stimulate the release of cytokines, alter vascular reactivity, induce inflammation and angiogenesis, and are involved in cancer metastasis [7,8]. It has been shown that PMP can express and transfer functional receptors from platelet membranes, such as glycoprotein IIb-IIIa (GPIIb-IIIa) and P-selectin, to different cell types [9] and, by that, enhance engraftment of hematopoietic stem/progenitor cells [10], as well as confer adhesive properties with circulating tumor cells (O. Dashevsky, E. Shai and D. Varon, unpublished data). In addition, PMP promote monocyte and neutrophil adhesion to the endothelium and stimulate COX-2 expression in monocytes and endothelial cells [11,12]. Furthermore, it was established that PMP could not only passively carry various proteins and receptors, but also chemoattract hematopoietic cells and stimulate their adhesion, survival, and proliferation [8]. Moreover, PMP © 2015 International Society on Thrombosis and Haemostasis
Platelets and their microparticles S41
activate intracellular signaling pathways such as ERK, PI3-kinase/Akt, and STAT proteins. These effects could only partially be overturned by heat and trypsin, suggesting that, in addition to intraparticle agents of protein nature, the lipid component of PMP could also be involved. The current report describes the role of platelets and PMP in immunity and inflammation, angiogenesis, tissue regeneration, and cancer progression. Immunity and inflammation Platelets have a broad repertoire of receptor molecules that enable them to recognize pathogen-associated molecular patterns and sense infection-induced inflammation. Different Toll-like receptors have been found on platelets. These receptors bind ligands from infectious pathogens and mediate microbial sensing and host defense mechanisms [13,14]. Platelets, along with their microparticles and with other inflammatory cells, can also facilitate microbial adhesion to endothelial cells (EC) in a variety of inflammatory diseases [15]. We have shown that in vitro treatment of EC with platelets significantly increases adherence of S. Aureus to the EC, both under static and moderate flow rate conditions [16]. These observations suggest a role for platelets in bacterial infection and colonization in the setting of endocarditis and other infections. More recent studies show the involvement of platelet factor 4 in bacteria-induced activation of platelets, through a common pathway that involves FccRIIA receptor that is present on the platelet surface. This interaction results in an inside-out signaling leading to integrin GPIIb-IIIa engagement and dense granule secretion [17]. Hu et al. [18] described the mechanisms of platelet interaction with monocytes, neutrophils, and lymphocytes, under static and flow conditions, suggesting different mechanisms for each of these interactions. Recently, it was demonstrated that recruited neutrophils scan for activated platelets through the selectin ligand P-selectin glycoligand-1 to initiate inflammation [19]. Although platelets exert antimicrobial effector mechanisms and initiate cross talk with the innate and adaptive immune factors, platelets may also induce injurious systemic responses in various infectious syndromes. Infection with dengue virus, for instance, may induce thrombocytopenia through two different mechanisms – impaired thrombopoiesis and peripheral platelet destruction via direct interaction [20]. Malaria is also associated with thrombocytopenia due to peripheral destruction, compromised platelet function and excessive sequestration of platelets in the spleen. During cerebral malaria, platelets were shown to be deleterious to the brain endothelium, by allowing binding between infected red blood cells and the endothelial cells [21]. However, other studies indicate a protective function for platelets in the early stages of erythrocytic infection, © 2015 International Society on Thrombosis and Haemostasis
by killing the parasite within the red cell. Deficient platelets and aspirin treatment reversed this effect [22]. Recently, it was demonstrated that activated platelets generate significant amounts of oxidized phospholipids, indicating that phospholipid oxidation is a regulated process of likely importance during physiological hemostasis and in acute response to injury. [23]. Oxidized low-density lipoproteins (ox-LDL) have an important role in the development of inflammatory-related vascular disease, such as coronary artery disease and atherosclerosis. We investigated the effects of homocysteine and ox-LDL on EC thrombogenicity. Treatment of EC with either homocysteine (1 or 10 mmol L1 for 16 h) or ox-LDL (100 lg mL1 for 16 h) resulted in a 2- to 3-fold enhancement in platelet adhesion under flow conditions at a shear rate of 350 s1. The enhancement in platelet adhesion induced by homocysteine was absolutely dependent on fibrin formation. Homocysteine treatment has significantly increased the cell surface TF activity, while oxLDL treatment upregulated intercellular adhesion molecule (ICAM)-1 expression and had no significant effect on endothelial TF activity. The homocysteine-induced enhancement in platelet adhesion was significantly reduced by blockade of the EC TF activity as well as by inhibition of the EC avb3 integrin. On the other hand, oxLDL-induced enhancement in platelet-EC adhesion was greatly inhibited by blocking ICAM-1 or avb3, but remained unaffected by inhibition of TF activity. Preincubation of platelets with the GPIIb-IIIa antagonist ReoPro virtually abolished the enhancing effect of both homocysteine and ox-LDL. These results suggest that homocysteine and ox-LDL might increase EC thrombogenicity by distinct mechanisms: homocysteine, by inducing TF activity; and oxLDL, by upregulating ICAM-1, both of which enhance GPIIb-IIIa/fibrinogen-dependent platelet adhesion to EC. The avb3 integrin, although not affected by EC stimulation, seems to play a crucial role in platelet-EC interaction regardless of the mechanism of EC perturbation [24]. Activated platelets have many mechanisms for delivering signals to target cells involved in inflammatory interactions. These diverse mechanisms include platelet secretion of mediators that affect local innate immune response both locally and through the secretion of PMP [25]. Platelets can also modulate adaptive immunity through their microparticles, at sites distant from the location of platelet activation [26]. Boilard [27] explored the mechanisms by which platelets are activated to release microparticles in the context of arthritis. Using genetically deficient mice and pharmacologic blockade, they showed that platelets form MP and participate in arthritis pathophysiology via stimulation of the collagen receptor GPVI. They further showed that other pathways such as thromboxane A2 stimulation, ligation of the P2Y12, or GPIb-IX receptor do not regulate platelet MP generation in inflammatory arthritis. MP
S42 D. Varon and E. Shai
formation from platelets can be induced by high sheer stress alone. However, interaction of blood cells with TNF-a-activated EC under flow conditions can lead to changes in the proportion of circulating MP [28]. More recently, it was shown that some PMP contain respiratory-competent mitochondria. These plateletsecreted mitochondria serve a substrate of phospholipase A2 IIA (that can also be secreted by platelets) to yield inflammatory mediators that promote leukocyte activation [29]. As suggested before, this study also shows further evidence that PMP are heterogeneous in their biological activities depending on the cargo they carry. Angiogenesis and tissue regeneration The role of PMP in blood vessel development was first shown by Kim [7]. They reported that PMP promote proliferation of EC. This effect was mediated by a concerted action of VEGF, FGF-2, and a lipid component of PMP, most likely sphingosine-1-phosphate. This lipid constituent seems to mediate also the anti-apoptotic and chemotactic effects of PMP on EC as well as PMP-induced tubule formation. We showed that platelets stimulate formation of blood vessels in vitro in the rat aortic ring model via VEGF and bFGF. Blockage of platelet factor-4 promoted this effect. Platelet releasate was found to induce EC chemotaxis, mediated by a concerted action of intraplatelet bFGF, PDGF, VEGF, and heparanase. A concomitant effect of bFGF and VEGF seemed to be essential for the entire process of vessel formation, while PDGF and heparanase were effective only at the migration stage. We concluded that platelets affect different stages of the angiogenic response with a trend to a pro-angiogenic net effect despite the presence of angiogenesis inhibitors [30]. The pro-angiogenic effect of platelets and PMP raises the question of what is the mechanism for this effect in view of the presence of angiogenic inhibitors (along with activators) in platelets a-granules. One potential explanation was offered by Italiano [31], who showed differential localization of angiogenic cytokines in different granules. This idea was further established by a different group who showed by electron tomography and 3D analysis that the a-granule population is morphologically heterogeneous. They visualized long, highly curved membrane strands, connected to spherical domains and confirmed that these tubules represent a-granules. They proposed that cargo is differentially released from tubular granules compared with spherical granules [32]. Jonnalagadda et al. showed that platelet secretion was kinetically heterogeneous in response to four different agonists. They found that the extent and rate of release are related to agonist potency with thrombin inducing the most rapid release of the highest number of cargo. However, cargo with opposing actions had similar release profiles, suggesting that platelet release can be calcified to
at least 3 classes of release processes differing in rate, but the distribution of cargo into each class is random [33]. It has been shown that PMP also differ in their content of exposed membrane anionic phospholipids and their intrinsic procoagulant potential, depending on the stimulant used to generate them [34]. We investigated the proteome of PMP produced by either thrombin stimulation or by shearing the platelets under arterial flow conditions (1800 s1). These studies revealed significant changes in the expression of several proteins related to cytoskeleton and platelet shape change machinery [35]. In a recent proteome analysis, Ve0 lez et al. [36] showed that the platelet secretome varies with the stimulus by comparing the platelet releasate following platelet activation with thrombin or collagen. These results are line with the studies that show that the release of angiogenesis regulatory proteins is modulated by physiological processes [31,37]. These studies demonstrated that activation of platelets with ADP stimulated the release of VEGF, but not endostatin, whereas thromboxane A2 released endostatin but not VEGF. They also showed that platelet releasates, generated by activation with ADP, promoted migration and formation EC tubules in in vitro angiogenesis models. We have demonstrated that PMP induce sprouting both in vitro and in vivo (Fig. 1) to a degree comparable with that of whole platelets [38]. Moreover, application of PMP may have a therapeutic potential. Intramyocardial injection of PMP markedly elevated the amount of novel capillaries developed in the heart muscle in the background of ischemia (LAD ligation in a rat model, Fig. 2). Thus, PMP may further support the platelet-induced angiogenic response operating relatively independently of platelets. Alternatively, PMP may be a tool or a mediator through which platelets exert their pro-angiogenic effect given that various released molecules traditionally considered as soluble are in fact bound to MP [39]. The influence of PMP on the angiogenic activity of endothelial progenitor cells (EPCs) was shown by Prokopi [40]. Endothelial tube formation was stimulated by conditioned medium of EPC cultures; this effect was attenuated by removing MPs from the conditioned medium by filtration, ultracentrifugation, or inhibition of the formation of the platelet GPIIb-IIIa integrin complex. This study also emphasized the misinterpretations that might arise from PMP-mediated exchange of antigens. They showed that mononuclear blood cells uptake platelet-derived microparticles and exchange marker proteins, replicating the phenotype of EPCs. This might have important implications for determination of progenitor cells for clinical trials in stem cell therapy. We further studied the potential effect of PMP in a stroke model. We hypothesized that tissue regeneration secondary to activation of endogenous neural stem cells (eNSC) can be enhanced by delivering platelets and PMP to the ischemic brain. To examine these potential therapeutic effects, we injected platelet poor plasma © 2015 International Society on Thrombosis and Haemostasis
Platelets and their microparticles S43
A
1
2
3
4
6
8
#
# ^
6
*
4
Infarction+PMP
2 0
0
20
30
50
PMP, µg mL–1 protein
100
VEGF + bFGF
Fig. 1. Platelet-derived microparticles induce angiogenesis in the aortic ring model. A. PMP in the indicated concentrations were added to the rings. After 7 days of incubation, the preparations were fixed, stained, and photographed. The following samples are shown: (1) negative control, (2) PMP 20 mg mL1, (3) PMP 30 mg mL1, (4) PMP 50 mg mL1, (5) PMP 100 mg mL1 and (6) VEGF 50 ng mL1 + bFGF 50 ng mL1. Representative experiment out of 7; bar 0.5 mm. B. Quantitative analysis of the pro-angiogenic effect of PMP. The experimental series were compared using unpaired Student’s t-test. *P < 0.02; vs. negative control (PMP 0), #P < 0.002 vs. negative control; ^P < 0.001 vs. negative control [38].
(PPP), FGF-2, PMP, and platelet lysate to the lateral ventricles after permanent middle cerebral artery occlusion (PMCAO) in rats. The animals were tested with the neurological severity score, and infarct volumes were measured at 90 days post-PMCAO. Immunohistochemistry was used to determine the fate of newborn cells and to count blood vessels in the ischemic brain. Platelets and PMP significantly increased eNSC proliferation and angiogenesis in the subventricular zone (SVZ) and in the perilesion cortex. Functional outcome was significantly improved, and injury size was significantly reduced in rats treated with platelet lysate suggesting additional neuroprotective effects (Fig. 3). Thus, a local delivery of platelet lysate or PMP to the lateral ventricles induces angiogenesis, neurogenesis, and neuroprotection and reduces behavioral deficits after brain ischemia [41,42]. Cancer progression The first report on the potential role of platelets in cancer progression descried the correlation between lung © 2015 International Society on Thrombosis and Haemostasis
200 160
P
