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Plasmodium falciparum infection is often associated with a procoagulant state. Recent identification of tissue factor (TF) in the brain endothelium of patients who died from cerebral malaria casts new light in our understanding of the coagulation disorder found in P. falciparum infection. It has also been revealed that parasitized red blood cells (pRBC) support assembly of multimolecular coagulation complexes. In this article, the role of TF expression by the endothelium and amplification of the coagulation cascade by pRBC and/or activated platelets—particularly at sequestration sites— is discussed as crucial events in mounting and sustaining a coagulation–inflammation cycle that contributes to organ dysfunction and coma in P. falciparum malaria.
Malaria caused by P. falciparum remains a highly endemic disease; it has been estimated that at least one million deaths occur every year. Children are at high risk, and often severe malaria (severe anemia or cerebral malaria [CM]) is the cause of death in this population 1–4. P. falciparum infection is associated with sequestration 5–8, endothelial cell (EC) activation 8–10, increase in inflammatory cytokines 3, 11, and activation of the coagulation cascade 12–20. Despite several papers devoted to the coagulation disorder in malaria—many of them published during the 1970s and 1980s (reviewed in Ref. 20)—the mechanism that triggers and propagates the coagulation activation in P. falciparum infection has remained elusive. These studies, however, demonstrated that bleeding and/or hemorrhage are typically not present, although laboratory tests indicate a certain degree of in vivo blood coagulation activation 12–20. Accordingly, prothrombin time (PT) and partial thromboplastin time (PTT) are close to normal values or prolonged, whereas thrombin-antithrombin complex (TAT) (which indicates in vivo thrombin formation) and D-dimers (which are a marker of in vivo fibrinolysis) are often elevated, and thrombocytopenia is a usual finding 12–21. This laboratory profile is compatible with disseminated intravascular coagulation (DIC), which is diagnosed according to a score defined by the International Society of Thrombosis and Haemostasis (ISTH)22. The scoring system takes into account i) platelet count, ii) elevated fibrin related markers, iii) prolonged prothrombin time, and iv) fibrinogen level 22 23. Depending on the score punctuation, levels < 5 are suggestive of non-overt (compensated) DIC while scores > or = 5 are compatible with overt (decompensated) DIC. Bleeding is more common in overt DIC, but it is not needed for the formal diagnosis 22 23. In other words, absence of bleeding in P. falciparum-infected patients does not exclude the presence of DIC in this same population; actually, most of these patients meet the criteria for DIC as defined above 22. Unfortunately, absence of bleeding is often mistakenly regarded as the reason why DIC is supposedly not present in malaria.
Further, some reports do not support the view that coagulation cascade/DIC play a role in malaria pathogenesis because fibrin is not always found in post-mortem studies. There has actually been studies in children* abstract and in adults where the presence of fibrin has been detected in post-mortem samples (reviewed in 20 ). While it is also correct that fibrin it not found in all autopsies, it cannot be excluded that fibrin has been formed sometime during the infection, and degraded by plasmin possibly generated by compensatory fibrinolysis 23. The main argument that supports this notion is the fact that the vast majority, perhaps all patients with mild and severe malaria, present elevated levels of D-dimers20, a specific marker of plasmin-mediated fibrin proteolysis in vivo 23. Finally, it has been reported that hemostatic alterations correlate with parasitemia, thrombocytopenia, and/or severity of P. falciparum infection in a number of studies; in addition, antiparasitic treatment halts the coagulation disorder and improves clinical outcome 12–20.Therefore, it is plausible that hemostatic alterations play a role in the disease progression and organ failure observed in malaria 16, 20.
More recently, it has been demonstrated that P. falciparum-parasitized RBC (pRBC) induce tissue factor (TF) expression in the microvascular EC in vitro, and support the assembly of multimolecular coagulation complexes 10. TF is the clotting initiator 24–26 and a structural member of the cytokine receptor family, which signifies the expansion of the adaptive immune system in vertebrates, indicating a close connection of the coagulation pathways with the host response to infection 27. Accordingly, TF has been increasingly recognized as the interface between coagulation and inflammation 27–32. Remarkably, the brain endothelium of P. falciparum-infected children who died from CM) and other causes display TF expression as determined by immunohistochemistry (IHC) studies using monoclonal anti-TF antibodies 10. These findings provide new insights in our understanding of the events associated with coagulation activation during P. falciparum infection and provide the experimental basis for the tissue factor model for malaria pathogenesis.
Previous models have tried to explain malaria pathogenesis including the ‘sequestration’ and the ‘cytokine’ hypotheses. The sequestration hypothesis suggests that, ‘the presence of infected erythrocytes is an essential event‘ 33. The cytokine hypothesis proposes that, ’while significant cerebral sequestration is often present and sometimes block vessels (with predictable sequelae), it is not essential for the onset of the cerebral manifestation of malaria‘ 34.
An alternative model for malaria pathogenesis, the ‘Tissue Factor Model’ (TFM) has been proposed 20, based on recently published findings, where TF has been identified in the EC exposed to pRBC in vitro and in the endothelium of patients who died from CM, and other causes 10. Late-stage pRBC support assembly of multimolecular coagulation complexes, which are crucial components of the amplification steps of the coagulation cascade 10. The TFM combines concepts evolved from both sequestration and cytokine hypotheses. In contrast to previous models, however, TFM places TF expression by the endothelium and the amplification of the coagulation cascade by pRBC and platelets—particularly at sequestration site—as crucial steps mounting and sustaining a coagulation–inflammation cycle that contributes to organ dysfunction in malaria. It also suggests that malaria is a disease with a strong vascular component, or a disease of the endothelial cells and microcirculation 20.
The TFM differs from the previous hypothesis in five respects:
A picture emerges where a local inflammatory reaction initiated by sequestration-associated endothelium TF expression and amplification by pRBC and activated platelets results in production of proinflammatory cytokines and coagulation factors that might act locally and systemically in a synergistic manner, fueling the inflammation–coagulation cycle 27–32. This cycle might be over amplified in the vessels of the brain, a tissue where the anticoagulant thrombomodulin is absent 24. As a result, neutrophil, lymphocyte, and platelet interactions with the capillary endothelium promotes EC injury, increased vascular permeability, vesiculation, and cellular apoptosis 27–32. Accordingly, an apoptosis–coagulation–inflammation imbalance could occur in falciparum malaria, and it is plausible that the vicious cycle of coagulation and inflammation 27–32, 51 operate and contribute to organ dysfunction, particularly in severe P. falciparum infections. It is important to recognize that nitric oxide (NO) availability may be impaired by hemolysis and hypoargininemia in both human 47 and mice 48 infected with Plasmodium sp.; it has also been reported that carbon monoxide (CO) suppresses the pathogenesis of experimental cerebral malaria 45. While pleiotropic mechanisms may explain the action of these gazes 45 47 48, it is documented that NO negatively modulates endothelium activation and platelet function 52 whereas CO inhibits the expression of pro-inflammatory genes (e.g., TF and TNF-α) and prevents inflammatory events in vivo 53. Finally, the finding that TF is potentially a crucial mediator of malaria pathogenesis suggests that therapeutics targeting TF and/or EC 54 could be useful in the treatment of malaria and/or its complications 4, 55.
Severe malaria is associated to a certain degree, with uncontrolled infection, systemic inflammation syndrome and cytokinemia, increased capillary permeability, hypoxia, acidosis, EC activation, microvascular coagulopathy, and multiorgan dysfunction. While much remains to be understood in malaria pathogenesis, the multiorgan dysfunction syndrome often associated with morbidity in malaria appears to be the result, at least in part, of the coagulation-inflammation cycle 1–4, 27–32.
The molecular mechanisms by which activation of coagulation-inflammation cycle is associated with metabolic stress and organ dysfunction have been comprehensively discussed, particularly in the context of sepsis pathogenesis 27–32. Of note, in both conditions activation of blood coagulation takes place3, 10. Usually, an underlying disorder (e.g. P. falciparum infection) could be accompanied by coagulation activation and increase in inflammatory cytokines. Coagulation factors (e.g. FVIIa, FXa, thrombin) are highly proinflammatory molecules through activation of PAR and through induction of cytokine expression. Cytokines, in turn, induce TF expression, leading to the perpetuation of the coagulation responses 27–32. Injection of activated coagulation factors (e.g., FVIIa) in healthy volunteers is accompanied by cytokine production (e.g., IL-6), while administration of cytokines (e.g., TNF-α) is associated with a procoagulant state 27–32. Further, coagulation activation and DIC has been well documented in patients with severe, sometimes untreatable bleeding, and the clinical/pathologic relevance is undisputable; however, major bleeding occurs in a minority of patients with DIC, as it is the case in malaria (reviewed in Ref. 20). More common is the occurrence of organ failure, but there is considerable debate about the extent that (micro)vascular fibrin deposition, as a consequence of DIC, contributes to disease pathogenesis. In other words, does coagulation activation have any functional/pathologic significance in malaria? Several lines of evidence support an important role of coagulation activation/DIC in the development of organ failure. Actually, extensive data have been reported on post-mortem findings of patients with DIC. These autopsy findings include diffuse bleeding at various sites, hemorrhagic necrosis of tissue and, in some cases, fibrin-platelet microthrombi in small blood vessels and sometimes associated with endothelial damage 27–32. Most important, experimental animal studies of DIC and clinical trials in humans show amelioration of hemostatic defects by various interventions targeting the coagulation cascade—it appears to improve organ failure, and, in most cases, to reduce mortality 27, 31, 40, 41. Clinical studies also support the notion of coagulation as an important denominator of clinical outcome, and DIC has been shown to be an independent predictor of mortality in patients with sepsis and severe trauma 27–32. Taking malaria pathology studies into account, intravascular fibrin formation is known to occur in vivo as estimated by increased levels of D-dimers in many patients infected with P. falciparum 12–20. Pathology studies have also detected fibrin (or thrombus) deposition in organs of some patients who died from falciparum malaria 20.abstract
It should be noted that development of EC injury and organ damage in the experimental model for sepsis seems to depend largely on a combination of factors and not in only one component (e.g., fibrin deposition only) associated with the coagulation-inflammation cycle 27–32. For example, reduced fibrinolysis and the effects of inflammatory mediators on the microvascular endothelium seem to be particularly important. Indeed, suppressed fibrinolysis is believed to be one of the most important components of organ dysfunction during DIC. Low levels of α2-antiplasmin-plasmin complex and high levels of plasminogen-activator inhibitor 1 (PAI-1) could therefore function as markers for aggravating disease 27–32. Notably, this coagulation profile has been reported in falciparum malaria in a study involving 76 patients 14. While much remains to be learned about the mechanism of organ dysfunction in sepsis and malaria, there appears to be sufficient evidence to conclude that the coagulation-inflammation cycle and DIC contribute, at least in part, to organ failure under distinct pathologic conditions.
This work was supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health. We thank Drs. José Marcos C. Ribeiro, Thomas E. Wellems, Robert W. Gwadz, and Kathyrin Zoon (NIAID/NIH) for continuous encouragement and support. We are grateful to Karl B. Seydel, Robson Q. Monteiro, Richard O. Whitten, Jerrold M. Ward, and Terrie E. Taylor for helpful discussions and immunohistochemistry assays. We thank NIAID intramural editor Brenda Rae Marshall for assistance.
The author was not able to include many references due to space constraints imposed by the Journal.