The cirrhotic platelet: Shedding light on an enigma
Authors: Stephen Caldwell, Ton Lisman
Journal: Hepatology (2016)

Potential conflict of interest: Nothing to report. See Article On Page 571 When first reading the article by Raparelli et al.1 in this issue of Hepatology, we came away with quite different perspectives, as we suspect many readers will depending on their own experience and background in this field. In a laborious series of experiments on human blood samples, the authors have explored the relationship between circulating gut‐derived endotoxin and evidence of platelet activation that could contribute to a relative hypercoagulable state in patients with cirrhosis and possibly to disease‐related complications and progression. In many ways, this study offers important insight into platelet function in the pathophysiology of cirrhosis‐related coagulopathy.2 From another perspective, the study points out the need for more research in this challenging area, further refinements in bench methodology, and dynamic studies that take into account the clinically unstable patient with cirrhosis. Most of those involved with clinical care and/or laboratory assessment of bleeding and clotting in liver disease patients are aware of the emergence of newer concepts of a rebalanced but fragile hemostatic system in cirrhosis.3 Hence, there is a broad consensus on the status of the coagulation system in cirrhosis (normo‐ to hypercoagulable in most published studies).4 However, the status of the other two components of hemostasis, platelet function and fibrinolysis,5 is the subject of ongoing clinical controversy. It should also be noted that although key studies in this field (including the Raparelli et al. study) have included and compared Child‐Turcotte‐Pugh class A, B, and C patients—the latter of which may justifiably include some patients with "decompensated" cirrhosis—most of the patients have been relatively stable judging from the narrow creatinine ranges and from exclusion of bacterial infection and clinical sepsis. Thus, none of these studies clearly excluded or focused on the recently recognized and distinct entity of acute on chronic liver failure (ACLF), a very unstable condition in which the hemostatic pathways are likely to be especially unstable. Historically, platelet hypofunction has been thought to contribute to cirrhotic bleeding. In their article, Raparelli et al. provide additional evidence for enhanced in vitro and in vivo platelet activity in patients with cirrhosis. The authors performed classical platelet aggregation studies using light transmission aggregometry in patients and control subjects in which platelet counts in the control subjects were adjusted to obtain the same platelet count in both groups. Because platelet aggregation studies are highly dependent on platelet count, this adjustment is mandatory for a meaningful comparison; however, as outlined by the authors, many prior studies did not account for the difference in platelet count between patients and control subjects. Raparelli et al. show that when platelets are weakly activated by a "subthreshold" concentration of activator, platelet aggregatory potential is substantially increased in patients with cirrhosis compared with control subjects. Using a higher activator concentration, an experimental setup employed by routine diagnostic laboratories, platelet aggregation was similar between patients and controls. An inhibitor of Toll‐like receptor 4, a receptor for lipopolysaccharide (LPS) present in platelets, inhibits platelet aggregation to both "subthreshold" and "threshold" concentrations of activator, whereas the inhibitor is without effect in healthy control subjects. In addition, the authors observed enhanced plasma levels of markers of in vivo platelet activation such as CD40L, and increased levels of platelet‐monocyte complexes, which are also indicative for enhanced in vivo platelet activation. Altogether, these data are compatible with a process in which LPS‐induced platelet sensitization results in enhanced in vivo platelet activation. From a critical perspective, one may also argue that the data are compatible with intrinsic platelet hypofunction that is "rescued" by the stimulatory effect of LPS, at least in the threshold model. Regardless, the Raparelli et al. study is a welcome and groundbreaking addition to this under‐studied aspect of cirrhotic coagulation dysfunction. The paucity of studies on platelet function in patients with cirrhosis is due in part to the many technical difficulties involved in performing and interpreting platelet assays in these complex patients. The data reported by Raparelli et al. are a step forward in this regard, because the authors adjusted aggregation studies for the platelet count and used two distinct doses of activator. Nevertheless, the adjustment for platelet count may be suboptimal, because each control sample was manipulated to obtain the same platelet count as that for a given patient sample. This matching procedure may have introduced bias, because only control samples were diluted with various amounts of buffer. In addition to issues with the platelet aggregation test, the interpretation of markers of in vivo platelet activation is complex, because these markers are cleared by the liver such that elevated levels may not reflect in vivo platelet activation, but rather reflect accumulation of these markers due to a lack of clearance.6 Reliable approaches to study platelet functionality in cirrhosis thus appear restricted to studies on platelets themselves, such as by platelet count–adjusted aggregometry,1 platelet count–adjusted studies of platelet adhesion and aggregation under conditions of flow,11 or flow cytometric analyses.7 Flow cytometry can both identify the in vivo platelet activation status (for example, by measurements of platelet‐monocyte aggregates as performed in the study by Raparelli et al., or by assessment of markers of platelet activation on the platelet itself) and platelet function after stimulation, which is (in contrast to aggregometry) independent of the platelet count within the sample. However, an analysis of the literature on unequivocal assays of platelet function reveals that there is no consensus (Table 1). Table 1 - Examples of Published Studies on In Vivo Activation and Ex Vivo Platelet Function in Patients with Cirrhosis Compared With Healthy Controls in Unbiased Experimental Set‐Ups Study Population Methodology In Vivo Activation Status Ex Vivo Activatability Comments Raparelli et al.1 Mixed Flow cytometry, platelet count–adjusted aggregation Increased number of platelet–monocyte aggregates Enhanced activation with subthreshold doses of activator Normal activation with higher dose of activator Potze et al.7 NASH and ASH cirrhosis Flow cytometry Normal p‐selectin exposure on resting platelets Slightly decreased p‐selectin exposure after activation Some numerical differences were not statistically significant Alkozai et al.8 Mixed Flow cytometry Normal p‐selectin exposure on resting platelets Decreased p‐selectin exposure after activation Sayed et al.9 Hepatitis B and C cirrhosis Flow cytometry Substantially increased p‐selectin exposure and platelet–monocyte aggregates on resting platelets Although statistically not significant, decreased platelet activation with increasing severity of disease Ogasawara et al.10 ASH and hepatitis C Flow cytometry Increased p‐selectin (ASH and hepatitis C) and PAC‐1 positivity (hepatitis C) on resting platelets PAC‐1 is an antibody specific to the activated form of αIIbβ3 Lisman et al.11 Mixed Adhesion to collagen and fibrinogen under flow at standardized platelet count and hematocrit Normal adhesion to both surfaces Adhesion to fibrinogen was nonsignificantly higher in patients; adhesion to collagen in the presence of thrombin was nonsignificantly lower in patients Panasiuk et al.12 Not specified Flow cytometry Increased P‐selectin expression, on resting platelets but similar platelet–monocyte aggregates Laffi et al.13 Mixed Platelet count‐adjusted platelet aggregation using washed platelets Substantially decreased aggregation and production of thromboxane A2 Abbreviations: ASH, alcoholic steatohepatitis; NASH, nonalcoholic steatohepatitis. Where does this challenging study fit into the emerging concepts of hypercoagulability in patients with cirrhosis, a group usually most clinically notable for difficult‐to‐control bleeding? Hyperactivity of circulating platelets in relatively stable cirrhosis has several potential implications that warrant further investigation. Detailed knowledge of the hemostatic balance/imbalance of cirrhosis may assist in targeted therapeutic strategies for prevention and treatment of both hemorrhagic and thrombotic complications. Because anticoagulant strategies may even delay decompensation,14 there is an urgent clinical need for a better understanding of cirrhotic hemostasis. On the other hand, so little is established in this field that we can expect the unexpected, such as the protective effect of some forms of platelet activation in experimental models of cholestatic liver injury.15 Thus, although it makes sense based on prior anticoagulation therapy trials that antiplatelet therapy (especially in the setting of circulating activated platelets) might hinder the process of parenchymal extinction (i.e., organ atrophy), a great deal of trial and error is likely needed before we can know how this story will play out. In conclusion, it is clear that this type of clinical investigation requires a meticulous and often laborious approach to improve our understanding of the many currents and counter‐currents that define the clinical course and optimal management in cirrhosis. It should also be restated that ultimately, these results must take into account the extraordinarily dynamic changes in the unstable ACLF patient often with ongoing infection, sepsis, and renal dysfunction. We are looking forward to additional work in this area to guide best practices for management. Author names in bold designate shared co‐first authorship.