Platelets and innate immunity

Although platelets are best known as primary mediators of hemostasis, this function intimately associates them with inflammatory processes, and it has been increasingly recognized that platelets play an active role in both innate and adaptive immunity. For example, platelet adhesive interactions with leukocytes and endothelial cells via P-selectin can lead to several pro-inflammatory events, including leukocyte rolling and activation, production of cytokine cascades, and recruitment of the leukocytes to sites of tissue damage. Superimposed on this, platelets express immunologically-related molecules such as CD40L and Toll-like receptors that have been shown to functionally modulate innate immunity. Furthermore, platelets themselves can interact with microorganisms, and several viruses have been shown to cross-react immunologically with platelet antigens. This review discusses the central role that platelets play in inflammation, linking them with varied pathological conditions, such as atherosclerosis, sepsis, and immune thrombocytopenic purpura, and suggests that platelets also act as primary mediators of our innate defences.     

Platelets and viruses: an ambivalent relationship

Thrombocytopenia is a frequent complication of viral infections providing evidence that interaction of platelets with viruses is an important pathophysiological phenomenon. Multiple mechanisms are involved depending on the nature of the viruses involved. These include immunological platelet destruction, inappropriate platelet activation and consumption, and impaired megakaryopoiesis. Viruses bind platelets through specific receptors and identified ligands, which lead to mutual alterations of both the platelet host and the viral aggressor. We have shown that HIV-1 viruses are internalized specifically in platelets and megakaryocytes, where they can be either sheltered, unaltered (with potential transfer of the viruses into target organs), or come in contact with platelet secretory products leading to virus destruction and facilitated platelet clearance. In this issue, we have reviewed the various pathways that platelets use in order to interact with viruses, HIV and others. This review also shows that more work is still needed to precisely identify platelet roles in viral infections, and to answer the challenge of viral safety in platelet transfusion.     

Dissociation between inhibition of phospholipid methylation and production of PAF-acether by rabbit platelets

Platelet-activating-factor (PAF-acether, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) is formed by and released from rabbit platelets stimulated with thrombin, with the ionophore A23187, with collagen and with the platelet-stimulating glycoprotein convulxin. We here show that 3-deazaadenosine (C3ado) and L-homocysteine (HCy), two inhibitors of platelet methylation, reduced the formation of PAF-acether and of its deacetylated product lyso-PAF-acether by rabbit platelets challenged with thrombin, under conditions where the accompanying aggregation was not significantly modified. In contrast, platelet aggregation induced by convulxin was completely and irreversibly blocked when C3ado and HCy were associated. Aggregation by thrombin was not affected by the methylation inhibitors even when ADP was scavenged and thromboxane formation was suppressed. Our results indicate that phospholipid methylation, thrombin-induced platelet aggregation and formation of PAF-acether can be dissociated. The formation of PAF-acether by rabbit platelets can be blocked by mechanisms other than inhibition of phospholipase A2, since the latter is not affected by C3ado and/or HCy.     

Dissociation between inhibition of phospholipid methylation and production of PAF-acether by rabbit platelets

Summary Platelet-activating-factor (PAF-acether, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) is formed by and released from rabbit platelets stimulated with thrombin, with the ionophore A23187, with collagen and with the platelet-stimulating glycoprotein convulxin. We here show that 3-deazaadenosine (C₃ado) and L-homocysteine (HCy), two inhibitors of platelet methylation, reduced the formation of PAF-acether and of its deacetylated product lyso-PAF-acether by rabbit platelets challenged with thrombin, under conditions where the accompanying aggregation was not significantly modified. In contrast, platelet aggregation induced by convulxin was completely and irreversibly blocked when C₃ado and HCy were associated. Aggregation by thrombin was not affected by the methylation inhibitors even when ADP was scavenged and rhromboxane formation was suppressed. Our results indicate that phospholipid methylation, thrombin-induced platelet aggregation and formation of PAF-acether can be dissociated. The formation of PAF-acether by rabbit platelets can be blocked by mechanisms other than inhibition of phospholipase A2, since the latter is not affected by C₃ado and/or HCy.     

Low-density lipoprotein and its effect on human blood platelets

Events leading to hyperactivity of human blood platelets are accompanied by an enhanced risk of atherosclerosis and arterial thrombosis. Lipoprotein disorders affect platelet functions, and hypersensitive platelets are observed in various stages of hyperlipidemia. Low-density lipoprotein (LDL), a circulating complex of lipids and proteins that is increased in hypercholesterolemia, enhances platelet function and increases sensitivity of platelets to several naturally occurring agonists. LDL sensitizes platelets via binding of apoB-100 to a receptor on the platelet membrane and via transfer of lipids to the platelet membrane. The receptor that mediates binding of LDL to the platelet and initiates subsequent intracellular signaling cascades has not yet been identified. Modification of native LDL generates a platelet-activating particle, and this interaction might contribute to the development of the atherosclerotic plaque. Lysophosphatidic acid is formed upon mild oxidation of LDL and is responsible for subsequent platelet activation induced by the modified LDL particle. Thus, LDL changes the functions of platelets via a broad spectrum of interactions.     

Platelet-derived chemokines: pathophysiology and therapeutic aspects

The identification of chemokines in blood platelets has strengthened our view of these cells as participants in immune host defense. Platelet chemokines representing prestored and rapidly releasable proteins may play a major role as first-line inflammatory mediators. This is evident from their capability to recruit early inflammatory cells such as neutrophil granulocytes and monocytes and even to exhibit direct antimicrobial activity. However, insight is growing that platelet chemokines may be also long-term regulators, e.g., by activating T lymphocytes, by modulating the formation of endothelium and even thrombocytopoiesis itself. This review deals with the individual and cooperative functionality of platelet chemokines, as well as their potential as a basis for therapeutic intervention in the pathology of inflammation, infection, allergy and tumors. Within this context, therapeutic strategies based on the use of antibodies, modified chemokines, chemokine-binding proteins and chemokine receptor antagonists as well as first clinical studies will be addressed.     

Integrins and cardiovascular disease

Cardiovascular diseases involve abnormal cell-cell interactions leading to the development of atherosclerotic plaque, which when ruptured causes massive platelet activation and thrombus formation. Parts of a loose thrombus may detach to form an embolus, blocking circulation at a more distant point. The integrins are a family of adhesive cell receptors interacting with adhesive proteins or with counterreceptors on other cells. There is now solid evidence that the major integrin on platelets, the fibrinogen receptor α _IIb  β ₃ , has an important role in several aspects of cardiovascular diseases and that its regulated inhibition leads to a reduction in incidence and mortality due to these disorders. The development of α _IIb  β ₃ inhibitors is an important strategy of many pharmaceutical companies which foresee a large market for the treatment of acute conditions in surgery, the symptoms of chronic conditions and, it is hoped, maybe even the successful prophylaxis of these conditions. Although all the associated problems have not been solved, the undoubted improvements in patient care resulting from the first of these treatments in the clinic have stimulated further research on the role of integrins on other vascular cells in these processes and in the search for new inhibitors. Both the development of specific inhibitors and of mice with specific integrin subunit genes ablated have contributed to a better understanding of the function of integrins in development of the cardiovascular system.     

Serotonin reuptake inhibitors and cardiovascular diseases: a platelet connection

Selective serotonin reuptake inhibitors (SSRIs) are a heterogeneous group of new antidepressants that cause a well documented acquired but reversible serotonin deficiency in blood platelets. Platelets are small, anucleate cells and are the only blood cells specialized in storing peripheral serotonin. Platelets are also an integral part of the hemostatic process that is initiated during pathologic thrombus formation in cardiovascular diseases. Serotonin release from platelets is important for functional hemostasis as indicated by congenital diseases with serotonin-deficient platelets that can lead to life-threatening bleeding problems. The postulate that SSRIs should have an impact on cardiovascular diseases is therefore well founded. Cardiovascular effects of SSRIs have indeed been shown in a number of studies investigating the effect of SSRIs in patients with psychosomatic comorbidity. SSRIs reduce the incidence of recurrent myocardial infarction (MI) in patients suffering from post-MI depression. In addition, SSRIs inhibit tight clot formation of platelets in vitro, which points to a direct anti-thrombotic or pro-fibrinolytic effect of SSRIs.Received 16 June 2004; received after revision 9 September 2004; accepted 23 September 2004     

Exogenous fibronectin modifies the aggregation of collagen-stimulated human platelets

Fibronectins (FN) are adhesive glycoproteins whose role in platelet aggregation is unclear. Addition of 3, 6 and 12 micrograms/ml of human plasma FN in vitro to isolated human platelets, which had been freed from plasma FN by gel filtration and subsequently stimulated with collagen, inhibited the last stage of platelet aggregation. With 3 and 6 micrograms/ml of FN a shortening of the lag-time was also observed. These data showed that FN may play a role in platelet-collagen interaction as well as in platelet-platelet interaction.     

The role of platelets in the pathogenesis of cerebral malaria

Malaria is a major cause of morbidity and mortality in the developing world and cerebral malaria is responsible for the majority of malaria-associated deaths. There is a strong association between thrombocytopenia and outcome in malaria, suggesting a role for platelets in the pathogenesis of malaria. This thrombocytopenia is likely due to platelet activation possibly through an interaction between PfEMP1 on plasmodium and CD36 on platelets. Platelet activation by plasmodium has two potential consequences. It can lead to the formation of micro-aggregates of infected red blood cells and platelets which can occlude blood vessels and it also leads to binding to and activation of the endothelium.     

Vascular integrins: pleiotropic adhesion and signaling molecules in vascular homeostasis and angiogenesis

New blood vessel formation, a process referred to as angiogenesis, is essential for embryonic development and for many physiological and pathological processes during postnatal life, including cancer progression. Endothelial cell adhesion molecules of the integrin family have emerged as critical mediators and regulators of angiogenesis and vascular homeostasis. Integrins provide the physical interaction with the extracellular matrix necessary for cell adhesion, migration and positioning, and induction of signaling events essential for cell survival, proliferation and differentiation. Antagonists of integrin alpha V beta 3 suppress angiogenesis in many experimental models and are currently tested in clinical trials for their therapeutic efficacy against angiogenesis-dependent diseases, including cancer. Furthermore, interfering with signaling pathways downstream of integrins results in suppression of angiogenesis and may have relevant therapeutic implications. In this article we review the role of integrins in endothelial cell function and angiogenesis. In the light of recent advances in the field, we will discuss their relevance as a therapeutic target to suppress tumor angiogenesis.     

Reelin is a platelet protein and functions as a positive regulator of platelet spreading on fibrinogen

Abnormalities of platelet functions have been linked to reelin-impaired neuronal disorders. However, little attention has been given to understanding the interplay between reelin and platelet. In this study, reelin was found to present in the human platelets and megakaryocyte-like leukemic cells. Reelin-binding assays revealed that extracellular reelin can interact with platelets through the receptor belonging to the low density lipoprotein receptor gene family. The reelin-to-platelet interactions enhance platelet spreading on fibrinogen concomitant with the augmentation of lamellipodia formation and F-actin bundling. In contrast, reelin has no effect on integrin αIIbβ3 activation and agonist-induced platelet aggregation. Molecular analysis revealed that the up-regulation of Rac1 activity and the inhibition of protein kinase C δ-Thr505 phosphorylation are important for reelin-mediated enhancement of platelet spreading on fibrinogen. These findings demonstrate for the first time that reelin is present in platelets and the reelin-to-platelet interactions play a novel role in platelet signaling and functions.     

Human endoglin as a potential new partner involved in platelet–endothelium interactions

Complex interactions between platelets and activated endothelium occur during the thrombo-inflammatory reaction at sites of vascular injuries and during vascular hemostasis. The endothelial receptor endoglin is involved in inflammation through integrin-mediated leukocyte adhesion and transmigration; and heterozygous mutations in the endoglin gene cause hereditary hemorrhagic telangiectasia type 1. This vascular disease is characterized by a bleeding tendency that is postulated to be a consequence of telangiectasia fragility rather than a platelet defect, since platelets display normal functions in vitro in this condition. Here, we hypothesize that endoglin may act as an adhesion molecule involved in the interaction between endothelial cells and platelets through integrin recognition. We find that the extracellular domain of human endoglin promotes specific platelet adhesion under static conditions and confers resistance of adherent platelets to detachment upon exposure to flow. Also, platelets adhere to confluent endothelial cells in an endoglin-mediated process. Remarkably, Chinese hamster ovary cells ectopically expressing the human αIIbβ3 integrin acquire the capacity to adhere to myoblast transfectants expressing human endoglin, whereas platelets from Glanzmann’s thrombasthenia patients lacking the αIIbβ3 integrin are defective for endoglin-dependent adhesion to endothelial cells. Furthermore, the bleeding time, but not the prothrombin time, is significantly prolonged in endoglin-haplodeficient (Eng ^+/?) mice compared to Eng ^+/+ animals. These results suggest a new role for endoglin in αIIbβ3 integrin-mediated adhesion of platelets to the endothelium, and may provide a better understanding on the basic cellular mechanisms involved in hemostasis and thrombo-inflammatory events.     

Platelet–bacterial interactions

Many bacteria are capable of interacting with platelets and inducing platelet aggregation. This interaction may be a direct interaction between a bacterial surface protein and a platelet receptor or may be an indirect interaction where plasma proteins bind to the bacterial surface and subsequently bind to a platelet receptor. However, these interactions usually do not trigger platelet activation as a secondary co-signal is also required. This is usually due to specific antibody bound to the bacteria interacting with FcγRIIa on the platelet surface. Secreted bacterial products such as gingipains and lipopolysaccharide may also be capable of triggering platelet activation.     

Activated scramblase and inhibited aminophospholipid translocase cause phosphatidylserine exposure in a distinct platelet fraction

Platelet procoagulant activity is mainly determined by the extent of surface-exposed phosphatidylserine (PS), controlled by the activity of aminophospholipid translocase and phospholipid scramblase. Here, we studied both transport activities in single platelets upon stimulation with various agonists. Besides the formation of procoagulant microparticles, the results show that a distinct fraction of the platelets exposes PS when stimulated. The extent of PS exposure in these platelet fractions was similar to that in platelets challenged with Ca²⁺-ionophore, where all cells exhibit maximal attainable PS exposure. The size of the PS-exposing fraction depends on the agonist and is proportional to the platelet procoagulant activity. Scramblase activity was observed only in the PS-exposing platelet fraction, whereas translocase activity was exclusively detectable in the fraction that did not expose PS. We conclude that, irrespective of the agonist, procoagulant platelets exhibit maximal surface exposure of PS by switching on scramblase and inhibiting translocase activity.Received 8 March 2005; received after revision 19 April 2005; accepted 13 May 2005     

L- and conventional forms of micrococci in the circulating blood of thrombocytopenic patients.

The multiplication of Gram-positive Cocci originating from L-forms carried by platelets of autoimmune thrombocytopenic patients, may be attributed to the primary platelet damage enhanced following interaction with bacteria.     

Prostacyclin-like activity of endothelium and subendothelium - important for atherosclerosis?

Morphological control of Moncada's bioassay for prostacyclin (PG I2) activity measurement shows that the activity depends not only on endothelium, but in important amounts on subendothelial tissue too. Therefore, it can be concluded that platelet thrombus formation after endothelial cell injury does not depend only on the PG I2-producing ability of the tissue.     

Platelets in defense against bacterial pathogens

Platelets interact with bacterial pathogens through a wide array of cellular and molecular mechanisms. The consequences of this interaction may significantly influence the balance between infection and immunity. On the one hand, recent data indicate that certain bacteria may be capable of exploiting these interactions to gain a virulence advantage. Indeed, certain bacterial pathogens appear to have evolved specific ways in which to subvert activated platelets. Hence, it is conceivable that some bacterial pathogens exploit platelet responses. On the other hand, platelets are now known to possess unambiguous structures and functions of host defense effector cells. Recent discoveries emphasize critical features enabling such functions, including expression of toll-like receptors that detect hallmark signals of bacterial infection, an array of microbicidal peptides, as well as other host defense molecules and functions. These concepts are consistent with increased risk and severity of bacterial infection as correlates of clinical abnormalities in platelet quantity and quality. In these respects, the molecular and cellular roles of platelets in host defense against bacterial pathogens are explored with attention on advances in platelet immunobiology.     

Platelet aggregation is inhibited by phycolectins

Lectins from four marine algal species were examined for interaction with human platelets. The lectin designated hypnin A, from the red algaHypnea japonica, inhibited adenosine diphosphate (ADP)-or collagen-induced human platelet aggregation in a dose-dependent manner. Complete inhibition was observed at concentrations of 100 and 5 g/ml of the lectin with, ADP (2 M) and collagen (0.2 g/ml)-induced platelet aggregation, respectively. At the inhibitory concentration of 0.5 to 100 g/ml, the lectin did not induce aggregation of resting platelets. Lectins from the other three algal species also inhibited ADP-induced human platelet aggregation. These results indicate that the algal lectins are a new group of inhibitors and may be useful to study glycoconjugates on platelet membranes and to design novel platelet aggregation inhibitors.