Preeclampsia: Platelet procoagulant membrane dynamics and critical biomarkers

Abstract

A state-of-the-art lecture titled “Preeclampsia and Platelet Procoagulant Membrane Dynamics” was presented at the International Society on Thrombosis and Haemostasis (ISTH) Congress in 2022. Platelet activation is involved in the pathophysiology of preeclampsia and contributes to the prothrombotic state of the disorder. Still, it remains unclear what mechanisms initiate and sustain platelet activation in preeclampsia and how platelets drive the thrombo-hemorrhagic abnormalities in preeclampsia. Here, we highlight our findings that platelets in preeclampsia are preactivated possibly by plasma procoagulant agonist(s) and overexpress facilitative glucose transporter-3 (GLUT3) in addition to GLUT1. Preeclampsia platelets are also partially degranulated, procoagulant, and proaggregatory and can circulate as microaggregates/microthrombi. However, in response to exposed subendothelial collagen, such as in injured vessels during cesarean sections, preeclampsia platelets are unable to mount a full procoagulant response, contributing to blood loss perioperatively. The overexpression of GLUT3 or GLUT1 may be monitored alone or in combination (GLUT1/GLUT3 ratio) as a biomarker for preeclampsia onset, phenotype, and progression. Studies to further understand the mediators of the platelet activation and procoagulant membrane dynamics in preeclampsia can reveal novel drug targets and suitable alternatives to aspirin for the management of prothrombotic tendencies in preeclampsia. Finally, we summarize relevant new data on this topic presented during the 2022 ISTH Congress.

Essentials

• •Preeclampsia is a hypertensive disorder in pregnancy that results in significant adverse maternal and neonatal outcomes.
• •A state-of-the-art lecture titled “Preeclampsia and Platelet Procoagulant Membrane Dynamics” was presented at the International Society on Thrombosis and Haemostasis (ISTH) Congress in 2022.
• •It remains unclear what mechanisms initiate platelet activation in preeclampsia.
• •However, facilitative glucose transporters 1 (GLUT1) and 3 (GLUT3) play a role in platelet activation and how platelets drive the thrombo-haemorrhagic abnormalities in preeclampsia.

1. Introduction

1.1. Preamble

Preeclampsia is a hypertensive disorder in pregnancy that results in significant adverse maternal and neonatal outcomes. Preeclampsia is characterized by the new onset of hypertension at or after 20 weeks gestation and is associated with proteinuria or other evidence of organ damage [1,2]. Gestational hypertension is distinguished from preeclampsia by the absence of proteinuria [[3], [4], [5], [6]], and preeclampsia exists on a spectrum of hypertensive disorders of pregnancy (HDP), which includes gestational hypertension and more severe disease such as early onset severe preeclampsia/eclampsia and hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome. There is significant heterogeneity in the clinical presentations of HDP, and the triggers that lead to the development of realized preeclampsia are multifactorial. This complexity is highlighted in research presented at the recent 2022 Congress of the International Society on Thrombosis and Haemostasis (ISTH).

1.2. The burden of preeclampsia

Preeclampsia affects 2% to 8% of all pregnancies and results in more than 70,000 maternal deaths and 500,000 fetal deaths worldwide every year [7]. It has become one of the most common indications for medically induced preterm birth and is the second leading cause of platelet count abnormalities after gestational thrombocytopenia, accounting for up to 16% of cases [8,9]. In addition, preeclampsia can lead to unpredictable clinical manifestations such as seizures, thrombosis, liver and renal failure, thrombotic microangiopathy, hemolysis, and bleeding complications [8,9]. Preeclampsia is associated with an increased morbidity risk to the mother (eg, hemorrhage, thrombosis, and liver failure) and can be associated with intrauterine growth restriction (IUGR), preterm delivery, and other neonatal comorbidities such as stroke, epilepsy, cardiovascular disease, and metabolic disorders later in childhood [8,9]. Preeclampsia mounts a significant economic burden on patients and the healthcare system, driven by higher rates of cesarean deliveries, maternal adverse events, high infant costs associated with preterm delivery, and infant adverse events [10]. Studies of maternal and infant healthcare costs related to preeclampsia have shown an estimated mean incremental cost of $28,603 per mother–infant pair compared with uncomplicated full-term healthy pregnancies and $17,608 compared with pregnant individuals with gestational hypertension [10].

1.3. Preeclampsia etiology: what we know

Pinpointing the exact initiators of preeclampsia and critical biomarkers to identify those at risk for developing the disease has been challenging. Part of the difficulty comes with the alterations in innate immunity, inflammatory, endothelial, and hemostatic responses that are naturally required for normal pregnancy to progress [[11], [12], [13], [14]]. Trophoblastic invasion, spiral artery formation, and endothelialization are critical for successful placental implantation and establishing the maternal–fetal circulation necessary for a viable and healthy pregnancy, although providing a barrier to protect the growing fetus against activated maternal host defenses [1,7,15]. When and how this natural process of placental implantation and formation becomes pathologic is key to understanding why preeclampsia develops. It is generally accepted that the placental disease and preeclampsia progresses in 2 stages [16]. First, there is abnormal placentation that occurs early in the first trimester, followed by a second stage of the “maternal syndrome” characterized by an excess of antiangiogenic factors and a thromboinflammatory response in the later second and third trimesters [[16], [17], [18], [19], [20]]. During the first stage, trophoblasts fail to adopt an endothelial phenotype. This leads to impaired trophoblast invasion and incomplete spiral artery remodeling. The resultant placental ischemia causes an increase in angiogenic markers such as soluble FMS-like tyrosine kinase 1 (sFLT-1) and soluble endoglin (sEng) [21,22]. sFLT-1 has been shown to bind and decrease levels of vascular endothelial growth factor (VEGF) and placental growth factor (PLGF) that are important mediators of endothelial cell function, especially in fenestrated endothelium of the brain, liver, and glomeruli [[23], [24], [25]]. This leads to an endothelial dysfunction in the maternal vasculature, which then triggers a multitude of pathogenic immune and inflammatory signaling and prothrombotic cellular activation resulting in the multiorgan systemic manifestations of preeclampsia [1,16,26].

1.4. Platelets’ role in preeclampsia pathogenesis and complications

Platelet activation and the complex interplay of platelets, vascular endothelium, and coagulation system activation is critical to the maternal thromboinflammatory response and possible adverse outcomes in preeclampsia [[1], [2], [3], [4], [5], [6]]. Changes in hematological parameters are common in pregnancy; for example, the incidence of thrombocytopenia ranges from 7.3% to 11.6% at delivery among women with uncomplicated pregnancies [27,28]. Although change in absolute platelet count is well characterized during pregnancy, less is known of platelet function during healthy pregnancies or preeclampsia [[29], [30], [31]]. Although platelet activation is well known to occur in preeclampsia, thrombotic or hemorrhagic sequelae remain unpredictable, and venous thromboembolism (VTE) rates can be 5× higher in preeclampsia postpartum, when compared with healthy pregnancies [32]. Paradoxically, preeclampsia patients are more likely to bleed at the time of labor and delivery [33]. Compared with normal pregnancies, patients with preeclampsia exhibit surrogate signs of heightened platelet activation including consumptive thrombocytopenia, increased mean platelet volume, generation of platelet microparticles, and expression of P-selectin and CD63 derived from platelet alpha and dense granule/lysosome, respectively [[34], [35], [36]]. Some of these platelet markers have been proposed as early predictors of preeclampsia and have been suggested to drive the prothrombotic state [[34], [35], [36]]. By contrast, other studies have shown impaired spontaneous and agonist-induced (ADP and collagen) platelet aggregation in platelets from patients with preeclampsia [[37], [38], [39], [40]]. Overall, preeclampsia platelets are dysfunctional; they are hyperactivated and prothrombotic yet less able to aggregate (Figure 1). The cause of this dysregulation of platelet function in preeclampsia is, until now, unknown. Taken together, it is likely that platelet activation is involved in the pathophysiology of preeclampsia and contributes to the prothrombotic state of the disorder. However, we do not know (1) what mechanisms initiate and sustain platelet activation in preeclampsia and (2) how platelets may be involved in the pathogenesis of hemostatic abnormalities in preeclampsia. By better understanding the mechanism of platelet activation, we can identify the causative and potential targetable mediators contributing to the different adverse outcomes in preeclampsia including early onset preeclampsia, HELLP syndrome, VTE, and bleeding risk.

Figure 1.

Platelet phenotypic duality in preeclampsia. Platelets in healthy pregnancy show signs of mild activation and priming, consistent with a prothrombotic state. In addition, healthy pregnancy platelets can mount full procoagulant response and form stable hemostatic plugs to limit blood loss after exposure of subendothelial collagen during caesarian sections [41,42]. Preeclampsia platelets, however, are preactivated in circulation, proaggregatory, partially degranulated, and may circulate as microaggregates/microthrombi [41,42]. Furthermore, the absolute procoagulant response of platelets in preeclampsia to subendothelial collagen is diminished, contributing to greater blood loss perioperatively [41,42]

1.5. Extracellular vesicles can trigger preeclampsia and is platelet dependent

Considerable efforts have gone into researching inflammatory initiators and its effects on trophoblastic differentiation that are important in the first stages of placental disease. The innate immune system naturally generates a protective response against signals of danger, not only against pathologic organisms but also against any challenges that may disturb the body’s homeostasis [12,13]. Physiologic changes in pregnancy disturb homeostasis in the host, and foreign DNA and proteins from the developing embryo trigger host immune defenses in the maternal circulation. The inflammasome is a tightly regulated supramolecular structure assembled in the cytoplasm of activated immune cells that lead to the proteolytic activation of proinflammatory caspases, which in turn drives subsequent systemic immune responses and related inflammation [43,44]. In the first stage of preeclampsia, inflammasome activation impairs trophoblast differentiation and proliferation, leading to endothelial dysfunction and a persistent inflammatory response, a hallmark of this placental disease. Kohli et al. [26] had previously shown that extracellular vesicles (EVs) are pathogenic and induces a preeclampsia phenotype in pregnant mice [26]. They also found that EVs caused activation and accumulation of maternal platelets within the placenta of preeclampsia mice, but not of control pregnant mice, and that aspirin and/or depletion of maternal platelets protected the mice from the EV-induced preeclampsia phenotype. They identified NLRP3 (nod-like receptor-pyrin-containing proteins) and IL-1β as markers of inflammasome activation and that EV-dependent inflammasome activation within trophoblast cells is augmented by platelets [45]. These findings establish the thromboinflammatory crosstalk at the maternal-embryonic interface. In an examination of the protective effect of low-molecular-weight heparin (LMWH), Kohli et al. [46] found that LMWH rescued EV-injected mice from embryonic cell death and preeclampsia. The authors showed that LMWH prevented EV-induced trophoblast inflammasome activation, restored altered trophoblast differentiation, and improved proliferation. Kohli et al. [46] also showed that LMWH activated the HB-EGF (heparin-binding epidermal growth factor) and PI3-kinase-AKT signaling pathways in trophoblast cells and prevented inflammasome activation. Both HB-EGF and PI3-kinase-AKT pathways are known mediators of vascular remodeling and angiogenesis [46]. The role of LMWH to prevent preeclampsia clinically is still unclear because combined patient level data from 8 randomized controlled trials showed no additional benefit of LMWH to prevent recurrent placenta-mediated pregnancy complications [47].

A protective effect on embryonic survival has been demonstrated for trophoblast thrombomodulin in a model of preeclampsia [48]. Thrombomodulin (TM) is a key anticoagulant on the surface of endothelial cells and binds with thrombin to accelerate protein C activation. Thrombomodulin is expressed on trophoblasts and syncytiotrophoblasts in the placenta. Pregnant transgenic mice with TM-null placenta result in embryonic lethality because of severe consumptive coagulopathy induced by the TM deficiency. Inflammasome inhibition (NLRP3-/- mice or Anakinra, IL-1R antagonist, and treatment) did not rescue the TM-null embryos from lethality and established that inflammasome activation is not the cause of embryonic death [48]. In addition, EV treatment reduced TM expression and trophoblastic proliferation, which could be rescued with Anakinra (inflammasome inhibition), and treatment with solulin (soluble, recombinant thrombomodulin) reduced EV-induced and platelet-mediated inflammasome activation (measured by NLRP3 and IL-1β expression), maintained placental TM expression, and reduced EV-induced fetal death in pregnant C57BL/6 mice injected with EVs [49].

The work by Kholi et al. [26,45,46,48] establishes the importance of the inflammasome in altering the trophoblast’s ability to establish the appropriate placentation and maternal–fetal homeostasis needed for a healthy pregnancy. The inflammasome may be the first step in triggering an overactive maternal immune response and inflammatory state that sets the stage for a subsequent complement, endothelial and platelet activation. This then perpetuates the inflammatory response leading to microthrombi and vascular dysfunction that characterizes the later stages of realized preeclampsia in the second and third trimesters. Both EVs, platelets, and phenotypic changes in activated platelets/platelet microparticles are important in this early process, and it is well established that platelets augment the inflammatory milieu generated by placental ischemia and contributes to the thrombohemorrhagic states and possible progression of the disorder. Therefore, research focused on the platelets’ role in preeclampsia etiology and pathophysiology may uncover new markers for predicting, diagnosing, and managing preeclampsia.

1.6. A method of platelet membrane procoagulation analysis

Standard coagulation tests such as prothrombin time or international normalized ratio, activated partial thromboplastin time (aPTT), and tests of plasma coagulation factors are not predictive of increased bleeding or thrombosis risks [50]. Compared with conventional in vitro platelet function tests that rely on surrogate measures of platelets’ ability to aggregate, advanced imaging study of platelet procoagulant membrane dynamics (PMD) directly examines platelet interactions and the spatiotemporal dynamics of the morphological and functional changes that platelets undergo during thrombosis and hemostasis [16,17]. Platelet PMD is a critical amplifier of the blood clotting process, and indicators of PMD are sensitive monitors of thrombosis and hemostasis [[51], [52], [53], [54], [55], [56]]. We described platelet PMD as the composite morphological changes that platelets undergo to amplify thrombin generation, including stimuli-induced activation, membrane protein expression, membrane blebbing, membrane ballooning, membrane phosphatidylserine (PS) exposure, breakdown of the membrane during microvesiculation, and the functional segregation of platelet phenotypes in a growing thrombus. Using fluorescently labeled indicators of platelet activation, granular secretions, oxidative stress, bioenergetics, and membrane procoagulation, coupled with high-resolution confocal and darkfield microscopy, we used the 3- or 4-dimensional imaging approach to quantify PMD at the single platelet and thrombi level [18,19]. Our platelet PMD analysis is systematic; it is an iterative approach that quantifies the intensity and spatiotemporal distribution of fluorescent dyes in high-resolution images of platelets and other blood cells over inert or procoagulant surfaces [[51], [52], [53], [54], [55],57,58]. Our PMD study can be combined with superresolution microscopy [57], transmission electron microscopy (TEM) [55,57], scanning electron microscopy (SEM) [54], and atomic force microscopy for state-of-the-art intracellular structure elucidation. To investigate cellular mechanisms and molecular mediators/drivers, we have coupled platelet PMD studies with flow cytometry [41], biochemical [55,59], and immunoassays [59] and to proteomic analysis [41,60]. We use Volocity and proprietary software for image deconvolution, quantification, 3- or 4-dimensional reconstruction, and movie rendering.

1.7. Detailed dynamic imaging of platelet activation in preeclampsia

To better understand the platelet behavior at the secretory and membrane levels, we conducted an observational study of 21 pregnant individuals in third trimester with preeclampsia, 10 pregnant individuals with gestational hypertension, 20 normotensive healthy pregnant individuals, and 19 nonpregnant female controls [41,42]. The study was presented in a state-of-the-art talk at the 2022 ISTH congress (London) [42]. In brief, we imaged platelets at high resolution in 4-dimensional space (ie, 3-dimensional in real time) and systematically analyzed platelet PMD. Characteristically, platelets derived from preeclampsia patients were preactivated and proaggregatory, and we visualized both homotypical and heterotypical aggregates significantly more in preeclampsia than in healthy pregnancy plasma and whole blood [41,42]. We confirmed that platelets of preeclamptic patients had objective features of hyperactivation (ie, increased filipodia) and were partially degranulated as fewer alpha-granules can be seen in TEM images, and granule content (determined by proteomics analysis) were depleted in comparison to healthy pregnancy [41,42]. Preeclampsia platelet membranes were procoagulant when compared with platelets in healthy pregnant patients, as determined by the quantification of membrane P-selectin and PS exposure and the formation of thrombin on the platelet membrane (Figure 1). Paradoxically, platelets of preeclamptic patients stimulated on collagen-coated surfaces showed impaired adhesion, aggregation, and formed smaller primary hemostatic plugs (Figures 1 and 2) [41,42].

Figure 2.

Platelet activation and GLUT3 overexpression in preeclampsia. Preeclampsia platelets are preactivated, proaggregatory, and prothrombotic in circulation. In response to exposed collagen such as in vessel injury during caesarian section, preeclampsia platelets showed diminished procoagulant and aggregatory response that likely contribute to perioperative bleeding. Source: Agbani E.O. Illustrated State-of-the-Art Capsules of the ISTH 2022 Congress [42].

1.8. Heightened membrane expression of facilitative glucose transporter-3 sustain platelet activation in preeclampsia

Previous studies have established the expression of facilitative glucose transporters 1 (GLUT1) and 3 (GLUT3) in human platelets. Although GLUT1 is reported to be located mainly on the platelet membrane, GLUT3 is reportedly localized majorly to alpha granule and is mobilized and secreted to the platelet membrane after activation [61,62]. Together, both transporters regulate glucose influx into platelets for basal metabolic demands and full procoagulant response after stimulation [[62], [63], [64], [65], [66], [67], [68], [69]]. The differential sizes of platelet glycogen storage seen in our TEM images of preeclampsia platelets in comparison with platelets in healthy pregnancy led us to visualize and quantify GLUT3 and GLUT1 expressions by our detailed imaging approach. We then determined that platelets in preeclampsia showed enhanced membrane expression of GLUT3, when compared with normotensive pregnant and nonpregnant women [41,42]. The GLUT3 expression in preeclampsia was seen in both unstimulated and stimulated platelets after adherence to and aggregation over collagen-coated surface.

The spatial distribution of GLUT3 in preeclampsia platelets is consistent with an active granular secretion and PMD, which suggests that preeclampsia platelets are constitutively activated and secrete alpha-granule contents in circulation [41,42]. Instead, of being quiescent and rested while in circulation, preeclampsia platelets are actively shape changing, secreting alpha-granule contents, aggregating, and forming circulating microthrombi [41,42]. We speculate that this high-energy state is supported by the increased expression of GLUT3 on the platelet membrane, which then provide the additional glucose influx required for the increased metabolic demands and sustained activation. Paradoxically, we also noted that preeclampsia platelets aggregation was significantly attenuated in response to collagen. Thus, when required to form a primary hemostatic plug, such as after injury and exposure of the subendothelial collagen during caesarian section, most preeclampsia platelets have lost the energetic burst and depleted the granule cargoes needed to aggregate and evoke a full procoagulant response [41,42] (Figures 1 and 2).

1.9. What initiates platelet activation in preeclampsia?

We do not know the precise agonist or combination of agonists activating circulating platelets in preeclampsia. Still, such add-on stimulation does not seem to be present in a healthy pregnancy. Our multiplex immunoassays confirmed previous reports of increases in plasma cytokines including SDF-1α, which can directly activate platelets [70] but also increases in i-309 and CTACK cytokines, whose effects on platelets are unknown. In a recent proteomic profiling of preeclamptic patients, we found 17 proteins to be significantly enriched in preeclampsia plasma [60]. Of these, the absolute log2-fold change indicated that S100A9 (S100A9), cadherin-5 (CDH5), caspase-12 (CASP12), fibronectin (FN1), and apolipoprotein E (APOE) were markedly elevated in preeclampsia [60]. Further research is now needed to resolve the role of these agonist(s) in the differential mobilization of GLUT1 and GLUT3 in the platelets of preeclamptic patients.

1.10. Predicting preeclampsia: urgent yet elusive

Predicting preeclampsia remains a diagnostic challenge because of the heterogeneity of its presentation [7]. The historical definition of preeclampsia as hypertension with new proteinuria is a poor predictor of adverse outcomes and traditional approaches to predict preeclampsia using maternal characteristics and history have low accuracy [7,71]. Still, there is a need for earlier and more accurate identification of pregnant patients at risk of preeclampsia and related adverse outcomes. Early and accurate identification of pregnancies at risk of preeclampsia is critical for early prevention and surveillance strategies. In addition, preeclampsia prevention with the current mainstay medication aspirin is only moderately effective; therefore, there is an urgent need to develop alternative therapeutics to treat and prevent preeclampsia in pregnant patients at risk. Studies have shown that the ratio of soluble FMS-like tyrosine kinase 1 to PLGF (sFLT1/PlGF) is highly predictive for the absence of preeclampsia in the short-term (within 4 weeks) but remains poor at ruling in preeclampsia (positive predictive value 36.7%) [6,72]. Similarly, multimarker screening tools (eg, Fetal Medicine Foundation UK algorithm) that incorporate historical factors, imaging measurements, and biomarkers have been validated to detect up to 90% of pregnancies that develop preterm (<37 weeks gestation) preeclampsia during first trimester screening [2]. However, predicting preeclampsia diagnosed at term remains limited, and early aspirin use does not change the rates of term preeclampsia.

1.11. Translational value of platelet GLUT3 overexpression in preeclampsia

Because GLUT3 is translocated to the platelet plasma membrane during secretion events [[67], [68], [69]], it is likely a marker of platelet activation but may have additional mechanistic contributions to sustaining pathologic platelet activation in preeclampsia by enhancing glucose uptake. Given that other markers of platelet activation, such as CD63, have been observed as early as the first trimester in women who developed preeclampsia, it is now important to characterize when GLUT3 overexpression occurs in pregnancy, especially in women at risk for preeclampsia, and how its expression changes relative to clinical phenotypes and adverse outcomes of preeclampsia. Together, platelet PMD and membrane GLUT3 expression may occur early and become exacerbated in pregnancies at high risk of preeclampsia and in those diagnosed with the disorder. The temporal expression pattern of GLUT3 or ratio expression of GLUT1 to GLUT3 (GLUT1/GLUT3) during pregnancy and preeclampsia development may be added biomarker for screening tools and to improve prediction algorithms for preeclampsia onset and progression. In addition, the selective inhibition of platelet GLUT3 may be a novel alternative therapeutic approach to antiprocoagulation and antithrombosis in preeclampsia.

1.12. Exploring potential biomarkers and triggers of preeclampsia complexities: insights from 2022 ISTH

Given the maternal–fetal morbidity and mortality associated with preeclampsia, there is significant interest in identifying early markers of endothelial dysfunction but also of immune, inflammatory, and procoagulant activation that are predictive of preeclampsia development and potential therapeutic targets for intervention. This is reflected in the preeclampsia research published in the 2022 ISTH abstracts.

1.12.1. Endothelial markers of preeclampsia

Some abstracts focus on the markers and mechanisms behind the early endothelial dysfunction in preeclampsia. For example, Nikolaeva et al. [73] demonstrates that endothelin-1 (ET-1) and endothelial microvesicles (EM) may be markers of recurrent severe preeclampsia. They showed that increased ET-1 and EVs are present at preconception in women with a history of pulmonary embolism (PE), who then develop recurrent PE. This points to the idea that preexisting or previous endothelial damage has primed the endothelium for additional damage leading to recurrent preeclampsia.

1.12.2. Complement markers of preeclampsia

EVs and activated platelets are not the only players that trigger inflammasome activation. The complement system, another important component of the innate immune system, can also trigger NLRP3 inflammasome activation through the deposition of the membrane attack complex on cell surfaces leading to increase intracellular Ca²⁺ concentrations that drive the assembly of NLRP3 inflammasome and caspase-1 activation [74]. Complement is not the only mediator of the inflammatory process but also there is crosstalk between the complement and coagulation cascades and the bidirectional ability of complement and platelets to activate each other. This has led to investigations not only in the role of complement, platelets, and prothrombotic markers as initiators of preeclampsia but also as markers of disease severity and progression of preeclampsia in the later stages of pregnancy. Under physiologic conditions, complement activation is important for the clearance of spent platelets and microparticles from the circulation in attempt to regulate their prothrombotic effects [75]. Fetal cells must evade recognition and activation of the complement system to achieve adequate trophoblast invasion and healthy placentation. In pathologic conditions, complement activation on or by platelets may contribute to thrombosis and thrombocytopenia, vascular dysfunction, and hemolysis, all of which can be seen in preeclampsia and related clinical syndromes (eg, HELLP, thrombotic thrombocytopenic purpura [TTP], and obstetrical antiphospholipid syndrome [APS]) [76]. Although there are different laboratory features that may be more predominant in the different microangiopathies, further research is still needed to better distinguish between these conditions because treatment differs.

1.12.3. Markers of coagulation activation in preeclampsia

Complement activation (C3a) excites platelets to express P-selectin resulting in further platelet activation allowing further complement fixation on the platelet surface and activation of the coagulation cascade and excess thrombin production. Complement can also activate tissue factor on monocytes, platelets, and other immune cells, enhancing procoagulant activity and inflammation. This conserved crosstalk of complement and coagulation cascades plays crucial roles in prothrombotic and inflammatory states such as preeclampsia. Abstracts at 2022 ISTH reported on markers of coagulation activation in preeclampsia as a possible early identifier of preeclampsia and risk for thrombosis. Nikolaeva et al. [77] demonstrated that early severe preeclampsia is characterized by increased expression to TF in the systemic circulation and decreased TF expression at local level (placental tissue). Tissue factor pathway inhibitor (TFPI) was lower in the plasma of preeclampsia patients. This supports the findings by Chinni et al., who showed that placentae from gestational vascular complications have significantly lower expression of TF, TFPI, TFPI-2, Anx V, and PAI-2 than those from the uncomplicated ones [78].

The intersection and reciprocity of complement, coagulation, and platelet activation is most evident in severe presentations of preeclampsia, such as HELLP syndrome, as well as obstetrical APS, and pregnancy-associated TTP. Neave et al. [79] found markedly increased sC5b-9 (complement) in preeclampsia/HELLP patients, compared with normal pregnancies, as well as increased VWF levels, decreased ADAMTS13 activity, and higher VWF antigen:ADAMTS13 ratios. In TTP, low ADAMTS13 levels (acquired or congenital) [80] result in reduced cleavage and inactivation of ultra-large VWF multimers, which then results in unregulated platelet activation, fibrin deposition, and microangiopathy causing platelet consumption, procoagulant activity, and hemolysis that can lead to both thrombotic and bleeding complications [79]. Abnormal complement and platelet activation in HELLP mirrors the processes seen in atypical hemolytic uremic syndrome and obstetrical APS where underlying disorders in factor H and MCP-1 predisposes to complement fixation on platelets and other immune cells resulting in hemolysis, thrombocytopenia, and thrombosis. In addition, the case reports of the successful use of eculizumab, a monoclonal antibody inhibitor of C5, in a patient presenting with preeclampsia/HELLP at 26 weeks and was able to stabilize maternal–fetal status, laboratory parameters, and improved the pregnancy outcome [81].

1.12.4. Platelet markers of preeclampsia

Platelet microvesicles appear to have some role in the abnormal placentation not only as an initiator of inflammasome activation and abnormal trophoblastic invasion but also as a propagator of the ongoing inflammation, complement activation, and consumptive coagulopathy that culminates in the maternal syndrome of preeclampsia later in pregnancy. There are multiple studies reporting on enhanced platelet activation in preeclampsia. Kurbanov et al. [82] looked at platelet aggregation patterns and intensity of aggregation in 112 pregnant women with preeclampsia compared with 50 healthy pregnant controls. This showed an increased intensity of not only irreversible platelet aggregation in women with preeclampsia but also hypoaggregation during stimulation with high-dose ADP [82]. Recently, we provided proteomic evidence that there are intrinsic abnormalities in the protein expression profile of platelets in preeclampsia that may identify those at risk for developing the maternal syndrome [60].

2. Conclusion And Future Directions

Several biomarkers of preeclampsia have now been identified. Because we do not yet know the cause of preeclampsia, it is difficult to confidently assign causality to any of these biomarkers. For the same reason, it is difficult to adopt these biomarkers for clinical practice. To validate possible biomarkers and better understand their role, large international prospective studies are needed to test multiple potential biomarker targets. Ideally, participants should be studied before pregnancy and followed throughout the 3 trimesters of pregnancy, and the research goals should focus on understanding the molecular mediators in preeclampsia etiology, in addition to predicting outcomes. Future research into preeclampsia biomarker should also focus on the validation of the several markers already identified in independent cohorts. To ensure the generalizability of the study outcomes across women of different races, both national and international collaborative approaches should be used. In vitro and animal models of preeclampsia are very useful approaches to accelerate our understanding of the human disease. Research should focus on the development of clinically relevant models of preeclampsia. This research should include evaluating standardized phenotypic features of preeclampsia to enable data pooling and provide a common ground for future preclinical and translational studies. In addition, it is important to recognize that much of the focus and understanding of preeclampsia pathophysiology pertains to early onset preeclampsia (preeclampsia developing before 34 weeks gestation). Early onset preeclampsia is associated with higher morbidity and mortality, especially for the fetus, compared with late-onset preeclampsia. Late-onset preeclampsia seems to be more associated with maternal characteristics rather than placental dysfunction because it is more often associated with normal placenta, fetal growth and birth weight [83]. Antiantigogenic markers such as sFLT1, PIGF, VEGFR, and endoglin (ENG) are less predictive of late-onset PE but more reliable for predicting early onset PE [84]. Thrombo-inflammatory pathways including platelet activation seems to still play an important role in the clinical manifestations of late-onset PE. More research distinguishing between the 2 categories is still needed.

Data from limited translational studies indicate that platelets’ role in preeclampsia is likely more in the progression and thrombo-hemorrhagic complications than in the cause of the disorder. Research is still needed to establish the precise plasma agonist and/or combination of agonist contributing to platelet activation in preeclampsia. The overexpression of GLUT3 or GLUT1 may be monitored alone or in combination (GLUT1/GLUT3 ratio) as a biomarker for preeclampsia onset, phenotype, and progression. Finally, controlling platelets’ role in preeclampsia with small molecule inhibition of platelets facilitative glucose transporters GLUT1 and GLUT3 may be problematic, given the evidence from the single and double knockout mouse models of GLUT1 and GLUT3 and the pivotal roles of these transporters for platelet metabolism and viability [[64], [65], [66]]. Notwithstanding, additional studies to further understand the signaling pathways and mediators of platelet activation and membrane procoagulation in preeclampsia may be able to reveal novel drug targets for clinically effective alternatives to aspirin to prevent, delay, and manage preeclampsia.