Discrepant Fibrinolytic Response in Plasma and Whole Blood during Experimental Endotoxemia in Healthy Volunteers

Background Sepsis induces early activation of coagulation and fibrinolysis followed by late fibrinolytic shutdown and progressive endothelial damage. The aim of the present study was to investigate and compare the functional hemostatic response in whole blood and plasma during experimental human endotoxemia by the platelet function analyzer, Multiplate and by standard and modified thrombelastography (TEG). Methods Prospective physiologic study of nine healthy male volunteers undergoing endotoxemia by means of a 4-hour infusion of E. coli lipopolysaccharide (LPS, 0.5 ng/kg/hour), with blood sampled at baseline and at 4 h and 6 h. Physiological and standard biochemical data and coagulation tests, TEG (whole blood: TEG, heparinase-TEG, Functional Fibrinogen; plasma: TEG±tissue-type plasminogen activator (tPA)) and Multiplate (TRAPtest, ADPtest, ASPItest, COLtest) were recorded. Mixed models with Tukey post hoc tests and correlations were applied. Results Endotoxemia induced acute SIRS with increased HR, temperature, WBC, CRP and procalcitonin and decreased blood pressure. It also induced a hemostatic response with platelet consumption and reduced APTT while INR increased (all p<0.05). Platelet aggregation decreased (all tests, p<0.05), whereas TEG whole blood clot firmness increased (G, p = 0.05). Furthermore, during endotoxemia (4 h), whole blood fibrinolysis increased (clot lysis time (CLT), p<0.001) and Functional Fibrinogen clot strength decreased (p = 0.049). After endotoxemia (6 h), whole blood fibrinolysis was reduced (CLT, p<0.05). In contrast to findings in whole blood, the plasma fibrin clot became progressively more resistant towards tPA-induced fibrinolysis at both 4 h and 6 h (p<0.001). Conclusions Endotoxemia induced a hemostatic response with reduced primary but enhanced secondary hemostasis, enhanced early fibrinolysis and fibrinogen consumption followed by downregulation of fibrinolysis, with a discrepant fibrinolytic response in plasma and whole blood. The finding that blood cells are critically involved in the vasculo-fibrinolytic response to acute inflammation is important given that disturbances in the vascular system contribute significantly to morbidity and mortality in critically ill patients.

Excessive sympathoadrenal activation is a hallmark of acute critical illness and the accompanying increase in circulating catecholamines induces widespread dose-dependent effects on metabolism and the vascular system [39][40][41]. Apparently, catecholamines induce opposite directed effects on the endothelium (progressive activation and damage) [42][43][44] and circulating blood (initial hypercoagulability followed by progressive hypocoagulability, hyperfibrinolysis and endogenous heparinization) [38,42,[45][46][47][48][49] and we infer that this reflects an evolutionary adapted response aiming at maintaining blood flow through a damaged and procoagulant microvasculature in the (shocked) critically ill patient [42]. Similarly to the opposite directed effects of catecholamines on the vascular endothelium and circulating blood [38,[42][43][44][45][46][47][48][49], the cells and fluid that constitutes the fluid phase of the vascular system i.e., the circulating blood, may also respond in opposite directions in acute inflammatory conditions in order to maintain homeostasis in the vascular system. In vitro studies of platelet function in sepsis and experimental endotoxemia have revealed that addition of septic plasma to control platelets can induce a pathologic response [28,50], and that removal of septic plasma from patient platelets can restore platelet function [28]. Also, fibrinolytic activity may both be enhanced and inhibited by circulating blood cells and cell-derived microparticles [51][52][53][54][55]. Thus, platelets protect the clot against tissue-type plasminogen activator (tPA)-induced fibrinolysis [51] and enhance the antifibrinolytic effect of exogenous FXIII in vitro [52] and red blood cells confer lytic resistance to fibrin resulting from modified fibrin structure and impaired plasminogen activation [56]. In contrast, other blood and endothelial cells, and their derived microparticles, promote fibrinolysis through the action of cellassociated urokinase-type plasminogen activator (uPA) and tPA [53][54][55] and leukocytes promote fibrinolysis through various alternative pathways [57]. Together, this emphasizes that platelets and other blood cells are critically involved in balancing the vasculo-fibrinolytic response, which may contribute to discrepant findings when evaluating fibrinolysis in plasma and whole blood. Given that sepsis and other acute inflammatory conditions are characterized by excessive changes in hemostasis and the vascular system that may be differently driven by and detected in whole blood and plasma, improved understanding and characterization of this response is of critical importance for optimizing and potentially goal-directing therapy.
The primary aim of the present study was to investigate and compare the functional hemostatic response in whole blood and plasma during experimental human endotoxemia as evaluated by Multiplate and by standard and modified TEG analyses, respectively. We expected that endotoxemia would induce a hemostatic response comparable to that previously described [23,24] and given the role of blood cells for fibrinolysis [51][52][53][54][55], we hypothesized that fibrinolytic activity during endotoxemia would differ in plasma and whole blood.
Here we report that experimental endotoxemia by means of a 4hour 0.5 ng/kg/hour LPS-infusion in healthy volunteers induced a hemostatic response with reduced primary but enhanced secondary hemostasis, enhanced early fibrinolysis and fibrinogen consumption followed by downregulation of fibrinolysis, with a discrepant fibrinolytic response in plasma and whole blood. Our findings indicate a critical role of platelets and/or other blood cells in the vasculo-fibrinolytic response to acute inflammation. The finding here that platelets and other blood cells, not present in plasma, may enhance fibrinolysis in acute inflammatory conditions is important given that especially thrombocytopenia is a strong predictor of poor outcome including excessive non-bleeding mortality in critically ill patients [58][59][60][61][62][63]. Since thrombocytopenia in septic patients often coexists with bleeding and excessive thrombus formation, or even overt disseminated intravascular coagulation (DIC) [59], it is tempting to speculate that thrombocytopenia may directly contribute to tip the hemostatic balance in the circulating blood towards a reduction in fibrinolysis and thereby paradoxically contribute to enhanced thrombus formation in the microvasculature.

Experimental human endotoxemia
The study was approved by the Scientific Ethical Committee of Copenhagen and Frederiksberg Municipalities, Denmark (file number H-A-2009-020 with amendments) and The Danish Data Protection Agency and was performed in accordance with the Declaration of Helsinki.
Nine healthy male volunteers (mean6SD age 2362 years) were enrolled after giving oral and written informed consent. All had an unremarkable medical history without signs of infection within 4 weeks ahead of the trial day and none took any regular medication. Before inclusion, volunteers underwent a thorough physical examination, a 12-lead electrocardiogram (ECG) was obtained and standard laboratory tests were performed. All tests were normal.
Endotoxemia: Following 12-hour overnight fast volunteers reported to the intensive care unit (ICU) at 7:30 am and were placed in bed. They were catheterized with an intravenous antecubital catheter (for LPS-infusion) and following local anesthesia (lidocaine, 20 mg/ml) an arterial line was inserted in the left radial artery. Heart rate (via a three-lead ECG), invasive blood pressure (mean arterial pressure, MAP) and peripheral oxygen saturation (SpO 2 , by pulse oximetry) were continuously monitored and medically qualified personnel were present at all times. Experimental endotoxemia was induced by means of a 4hour continuous intravenous infusion of purified Escherichia coli LPS (infusion rate 0.5 ng/kg/hour; Batch G2 B274, US Pharmacopeial Convention, Rockville, Maryland, US). In this model, plasma tumor necrosis factor (TNF)-a reaches its peak value at approximately 1 hour after cessation of the infusion [3,64]. As part of another sub-study, which investigated cerebral auto regulation (data reported elsewhere), noradrenaline was infused immediately after blood samples had been collected at baseline and 4 h as previously described [65]. Given the short in vivo half-life of noradrenaline (tK = 2.5 min in healthy individuals [66]) this drug could be assumed to be completely metabolized at the following blood sample time-points.
Blood was sampled for routine biochemistry and functional hemostatic measurements (TEG, Multiplate) at 0 h, 4 h and 6 h where 0 h denotes the time when LPS-infusion was initiated; thus 4 h corresponds to the cessation of the infusion (denoted ''during endotoxemia'') and 6 h is two hours after cessation. Volunteers were allowed to drink tap water ad libitum during the study day. They were discharged after 12 hours following removal of catheters and a light meal. According to the Danish Legislation, volunteers were not allowed payment alone for their attendance in the study. However, they were compensated for the loss of earnings and for any pain and suffering associated with the study.

Thrombelastography (TEG)
TEG whole blood clot formation was evaluated simultaneously in 3.2% citrated blood samples by kaolin-activated (TEG), kaolinheparinase-activated (heparinase-TEG) and tissue-factor (TF)activated platelet-blocked (TEG Functional FibrinogenH, FF) analyses by a TEGH 5000 Hemostasis Analyzer System (Haemonetics Corp., MA, US), according to the manufacturers recommendations. All analyses were conducted at 37uC. The simultaneous TEG and heparinase-TEG analysis allowed investigation of endogenous heparinization as previously described [38] [7]. To estimate platelet contribution to clot strength (MA) we calculated platelet MA (mm) by subtracting FF MA from TEG MA, with results reported as crude platelet MA and platelet MA per platelet (mm/10 9 platelets, platelet MA divided by platelet count).
The day-to-day coefficient of variation of whole blood TEG MA is ,7% in our laboratory [67].
In addition, the clotting potential of citrated plasma i.e., the capacity for formation of a pure fibrin clot was investigated by TEGH. Briefly, citrated plasma samples were thawed from 280uC just before analysis and 340 ml plasma was recalcified (20 ml 0.2 M CaCl 2 , final concentration 11.1 mM) and activated with TF (lipidated recombinant human TF, Innovin, Dade Behring, Marburg, Germany; final dilution 1:42,500) and analyzed immediately at 37uC. To assess the clot resistance to fibrinolysis, citrated plasma samples were analyzed with or without addition of 1.8 nM tissue-type plasminogen activator (tPA, single-chain, American Diagnostica, Greenwich, US) as previously described [67]. The TEG variables described above were recorded.

Statistics
Statistical analysis was performed using SAS 9.1 (SAS Institute Inc., Cary, NC, US). Data from volunteers were investigated by repeated-measures (RM) analyses (PROC MIXED, autoregressive covariance structure, SAS) and Tukey post hoc tests. Goodness of fit of the mixed model was assessed by investigating the residuals. Correlations between variables were investigated by Pearson correlations, and reported by R and p-values. Data are presented as means 6SD. P-values ,0.05 were considered significant.

Physiology, inflammation and routine coagulation tests
Endotoxemia induced a systemic inflammatory response syndrome (SIRS) as indicated by increased HR, temperature and white blood cell count and decreased MAP (Table 1). Furthermore, lymphocyte and monocyte counts decreased while CRP and procalcitonin increased. With regard to routine coagulation tests, platelet count, APTT and factor II, VII and X decreased and INR increased whereas plasma fibrinogen, D-dimer and antithrombin did not change (Table 1).

Hemostatic response in whole blood
Endotoxemia induced a decline in primary hemostasis (platelet adhesion and aggregation) as evaluated by MultiplateH TRAPtest, ADPtest, ASPItest and COLtest and it significantly reduced the per platelet response in the TRAPtest (Table 2).
Secondary hemostasis (fibrin-platelet clot formation in whole blood) also changed during and after endotoxemia as evaluated by standard and modified TEGH analysis (representative profiles displayed in Figure 1): TEG R-time decreased, G increased and CLT decreased during endotoxemia, indicating combined increases in coagulation factor activity, clot strength and clot lysis, respectively. In contrast, Functional Fibrinogen (whole blood, blocked platelet GPIIb/IIIa receptors) MA decreased after endotoxemia indicating reduced fibrinogen level and fibrin clot strength.
The calculated platelet contribution to TEG MA (platelet MA) increased during endotoxemia (p = 0.006) from 4165 mm at baseline (0 h) to 4765 mm at 4 h and 4662 mm at 6 h (both p,0.05 compared to 0 h), probably explaining the discrepant increase in TEG MA and decline in FF MA (Table 2). Since platelet count decreased during endotoxemia, the platelet MA per platelet also increased (p,0.001) from 0.1960.04 mm/10 9 platelets at baseline (0 h) to 0.2560.04 mm/10 9 platelets at 4 h and 0.2760.05 mm/10 9 platelets at 6 h (both p,0.001 compared to 0 h).
There was no evidence of endogenous heparinization as evaluated by the difference between TEG and heparinase-TEG (TEG minus heparinase-TEG) [38]: delta R at 4 h 20.160.

Hemostatic response in plasma
In contrast to the findings in whole blood, TEG Angle increased and TEG+tPA MA and G increased in plasma during and after endotoxemia indicating increased clot velocity and strength/ firmness, respectively (Table 2). Furthermore, plasma TEG+tPA Ly30/60 decreased considerably whereas CLT increased during and after endotoxemia, indicating increased resistance towards the tPA-induced in vitro clot lysis ( Figure 1 and Table 2).

Correlations between plasma fibrinogen, platelet count and TEG MA
The main determinants of TEG/ROTEM clot strength are fibrinogen and platelets (in addition to FXIII which we did not measure) [68][69][70][71]; we therefore investigated correlations between these variables and TEG MA during endotoxemia. At baseline, fibrinogen correlated significantly positively with TEG MA (r = 0.83, p = 0.006) whereas platelets did not (r = 0.14, p = NS). However, during (4 h) and after (6 h) endotoxemia, the contribution of fibrinogen to TEG MA decreased and became nonsignificant (r = 0.32 and r = 0.25, both p = NS) whereas the contribution of platelets to TEG MA increased non-significantly at 4 h (r = 0.55, p = 0.128), indicating increased platelet reactivity in the context of declining platelet count. After endotoxemia (6 h), TEG MA and platelet count did not correlate significantly (r = 0.18, p = NS).

Discussion
The main finding of the present study was that experimental endotoxemia, along with induction of SIRS, resulted in a  hemostatic response in whole blood with reduced primary but enhanced secondary hemostasis, enhanced early fibrinolysis and fibrinogen consumption followed by inhibition of fibrinolysis. In plasma, however, endotoxemia resulted in a progressive increase in clot resistance towards tPA-induced fibrinolysis indicating that the early LPS/inflammation-induced increase in fibrinolytic activity may be mediated by cellular elements in the whole blood. Sepsis is associated with vascular dysfunction and coagulopathy that may progress from an initially normal coagulation profile to hypercoagulability, hyperfibrinolysis and ultimately hypocoagulability with increasing disease severity [11][12][13]18]. This has been documented by TEG/ROTEM and has been reproduced in studies using experimental human [23] and animal [25][26][27] models of endotoxemia [23,[25][26][27]. Also, platelet aggregation is profoundly reduced in septic patients [19][20][21][22] and decreases with disease severity [20][21][22], which has also been reproduced in experimental human [23,24] and animal [28,29] models of endotoxemia.
In the present study, experimental endotoxemia, by means of a 4-hour infusion of 0.5 ng/kg/hour LPS, reduced primary hemostasis (platelet aggregation, reduced by Multiplate) and enhanced secondary hemostasis (clot formation) in whole blood (reduced R time (enhanced coagulation initiation) and increased G (enhanced clot strength) at 4 h by TEG) in accordance with previous findings [23][24][25][27][28][29]. During endotoxemia, fibrinolysis was enhanced (increased breakdown of the platelet-fibrin clot, CLT reduced at 4 h by TEG), which probably explains the observed reduction in the functional fibrinogen level (reduced strength of the fibrin clot, MA reduced at 4 h by FF). However, after endotoxemia, fibrinolysis was inhibited (increased CLT at 6 h by TEG), in accordance with previous findings in human [23] and animal [25] studies of experimental endotoxemia.
In contrast to the reduction in platelet aggregation, platelet clot formation increased (the pure contribution of platelets to TEG clot strength, platelet MA increased at 4 h and 6 h) both in total and on a per platelet basis (i.e. each platelet became more reactive), in accordance with the notion that the platelet fibrinogen receptor activity (GPIIb/IIIa) is enhanced in early sepsis [50].
Given that thrombosis may represent a critical component of innate immunity [72], the hemostatic changes observed in response to endotoxemia probably reflect part of a coordinated immune response. Thus, there is emerging evidence that platelet function goes far beyond hemostasis and that platelets are critically involved in and modulators of host defense and immune function [73][74][75][76]. Hence, Yaguchi and colleagues [21] suggested almost 10 years ago that sepsis induces a redistribution of platelet function from hemostasis toward other functions like e.g. vascular healing. We recently reported of an association between excessive sympathoadrenal activation and reduced hemostatic function of platelets in trauma patients [77,78] suggesting that catecholamines may promote a switch in platelet function from hemostasis to e.g. immunomodulation [42,76]. We infer that the change in hemostatic platelet function in critical illness may serve to sustain platelet circulation (avoiding consumption), thereby allowing platelets to exert other functions like e.g., vascular healing [21] and immunomodulation [76].
The oppositely directed responses in primary and secondary hemostasis observed in the present study probably reflect that we investigated an early hemostatic response to acute inflammation, in which it is well documented that secondary hemostasis is enhanced rather than reduced [11][12][13]23,25,27].
In contrast to the early increase (4 h) in and later downregulation (6 h) of fibrinolysis observed in whole blood by TEG, the resistance of the fibrin clot towards tPA-induced fibrinolysis increased progressively both during (4 h) and after (6 h) endotoxemia. This discrepancy indicates that blood cells and/or blood cell-derived microparticles and/or molecules enhance fibrinolysis in acute inflammation in accordance with the notion that inflammatory and endothelial cells and their derived microparticles can promote fibrinolysis [53][54][55]. Though platelets in some studies have been reported to enhance clot resistance towards fibrinolysis [51] it cannot be excluded that platelets under conditions characterized by systemic inflammation accompanied by endothelial activation and damage may change their phenotype towards a more pro-fibrinolytic one to avoid clot formation in the microvasculature thereby ensuring adequate organ perfusion. Given that degranulation of platelets is a highly regulated process [79,80], it is also tempting to speculate that thrombocytopenia, depending on the context, may both enhance and reduce fibrinolysis. Besides platelets, other blood cells, leukocytes in particular, may also through mechanisms such as release of neutrophil elasase, enhance fibrinolysis and thereby contribute to a discrepant response in plasma and whole blood [57]. Finally, this discrepancy emphasizes the importance of evaluating hemostasis in whole blood, opposite plasma, while also taking the concurrent state of the vascular endothelium (pro-vs. anticoagulant) into account since the states of the fluid (circulating blood) and solid (endothelium) phases of the vascular system may, from a systems biology perspective, counterbalance each other [42]. Thus, in conditions with systemic inflammation and/or coagulation activation, progressive hypocoagulability and fibrinolysis of the circulating blood may serve to keep a progressively more damaged and procoagulant microvasculature open in order to maintain perfusion of critical organs [42]. This notion is in accordance with the progressive hypocoagulability and fibrinolysis reported in septic patients [11][12][13]18] and it also agrees with the finding of hyperfibrinolysis in trauma [77,[81][82][83][84][85][86][87][88][89][90], cardiac arrest [91] and major surgical [92] patients and with the finding of enhanced protein C activation in patients with septic shock [93], during the reperfusion phase after cardio pulmonary bypass [94] and in cardiac arrest [95] or severely injured [77] patients. Despite thrombosis being a critical player in innate immunity [72], it is notable that several of the endothelial derived molecules that promote hypocoagulability exert potent antiinflammatory and cytoprotective functions [96][97][98][99] that may ultimately generate at survival advantage in critically ill patients.
We found no evidence to suggest that endotoxemia induces endogenous heparinization. This contrasts previous findings in septic [32][33][34][35][36][37] and trauma [38] patients. This discrepancy is likely explained by the presence of endothelial damage and hence glycocalyx degradation in patients with severe sepsis [5,6,100], which cannot be reproduced in human experimental endotoxemia since the LPS-doses required to mimic severe sepsis with regard to endothelial damage and/or organ dysfunction are unsafe and ethically unacceptable [1][2][3].
The present study has several limitations. Firstly, only young male volunteers were investigated in the experimental part and consequently a possible age and/or gender difference in response to endotoxemia was not evaluated. Furthermore, the low number of volunteers investigated increases the risk of introducing both type I and II errors, emphasizing that the findings herein should be confirmed in a larger studies. Finally, though the administered noradrenaline infusion [65] was assumed completely metabolized before blood sampling [66], it cannot be excluded that the infused noradrenaline influenced platelet number and function as catecholamines including noradrenaline influence both platelet adhesion and activation [45].
In conclusion, induction of experimental endotoxemia by means of a 4-hour LPS-infusion at 0.5 ng/kg/hour induced a hemostatic response comparable to that observed in early sepsis, with reduced primary but enhanced secondary hemostasis, enhanced early fibrinolysis and fibrinogen consumption followed by downregulation of fibrinolysis. In plasma, however, endotoxemia resulted in a progressive increase in clot resistance towards tPA-induced fibrinolysis. We infer that the discrepant fibrinolytic response observed in plasma and whole blood reflects a critical role of platelets and/or other blood cells in the vasculo-fibrinolytic response to acute inflammation in accordance with the finding here of significantly altered platelet function in response to endotoxemia.