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. Author manuscript; available in PMC: 2014 Nov 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2013 Sep 19;33(11):2529–2537. doi: 10.1161/ATVBAHA.113.302339

A role for platelet glycoprotein Ib-IX and effects of its inhibition in endotoxemia-induced thrombosis, thrombocytopenia and mortality

Hong Yin 1,*, Aleksandra Stojanovic 1,*, Weidong Xu 1, Adam Corken 4, Alexander Zakharov 1, Feng Qian 1, Sasha Pavlovic 3, Aleksandar Krbanjevic 1, Alexander V Lyubimov 1, Zaijie J Wang 2, Jerry Ware 4, Xiaoping Du 1
PMCID: PMC4043446  NIHMSID: NIHMS574833  PMID: 24051142

Abstract

Objective

Poor prognosis of sepsis is associated with bacterial lipopolysaccharide (LPS)-induced intravascular inflammation, microvascular thrombosis, thrombocytopenia, and disseminated intravascular coagulation. Platelets are critical for thrombosis, and there have been increasing evidence of the importance of platelets in endotoxemia. The platelet adhesion receptor, the glycoprotein Ib-IX complex (GPIb-IX), mediates platelet adhesion to inflammatory vascular endothelium and exposed subendothelium. Thus, we have investigated the role of GPIb-IX in LPS-induced platelet adhesion, thrombosis and thrombocytopenia.

Approach and Results

LPS-induced mortality is significantly decreased in mice expressing a functionally deficient mutant of GPIbα. Furthermore, we have developed a micellar peptide inhibitor, MPαC, which selectively inhibits the VWF-binding function of GPIb-IX and GPIb-IX-mediated platelet adhesion under flow without affecting GPIb-IX-independent platelet activation. MPαC inhibits platelet adhesion to LPS-stimulated endothelial cells in vitro and alleviates LPS-induced thrombosis in glomeruli in mice. Importantly, MPαC reduces mortality in LPS-challenged mice, suggesting a protective effect of this inhibitor during endotoxemia. Interestingly, MPαC, but not the integrin antagonist, Integrilin, alleviated LPS-induced thrombocytopenia.

Conclusion

These data indicate an important role for the platelet adhesion receptor GPIb-IX in LPS-induced thrombosis and thrombocytopenia, and suggest the potential of targeting GPIb as an anti-platelet strategy in managing endotoxemia.

Sepsis is a life-threatening systemic inflammatory state induced by the entry of bacterial endotoxins (such as lipopolysaccharides, LPS) into the blood circulation (endotoxemia). Mortality in severe sepsis is often associated with LPS-induced intravascular inflammation and thrombosis in microvasculature, which leads to impaired microcirculation, multiple organ failure, disseminated intravascular coagulation, and circulation failure (septic shock)1, 2. It is known that in order to improve the survival probability of septic patients, it is necessary not only to treat the source of infection, but also to treat systemic inflammation and microvascular thrombosis induced by endotoxemia and to improve the microcirculation.

There are two interrelated mechanisms causing thrombosis: (1) the coagulation cascade that catalyzes conversion of soluble fibrinogen in blood into a clot, and (2) platelet adhesion and aggregation, which not only form platelet thrombi, but also facilitate coagulation and inflammation. An anti-coagulant agent, the activated protein C (commercial name Xigris), was studied in clinical trials for its efficacy in the management of microvascular thrombosis and inflammation in adult sepsis. However, significant adverse effect of bleeding and associated mortality out-weighted the beneficial effect of the drug3, 4. It remains unclear regarding the exact roles of platelets in LPS-induced inflammation and microvascular thrombosis and whether platelet adhesion and activation contributes to mortality in endotoxemia patients. However, there is increasing evidence suggesting the importance of platelets in endotoxemia: LPS sensitizes platelets to agonist stimulation5, induces the accumulation of platelets in the blood vessels of certain organs such as the lung and liver6, 7, and induces consumptive thrombocytopenia6, 8, 9, which is associated with poor outcome of septic patients.

In the microcirculation, particularly arterioles wherein blood flows at relatively high shear rates, platelet adhesion to the vascular wall is dependent upon the interaction between GPIb-IX and its ligand, von Willebrand factor (VWF)10–12. GPIb-IX has also been shown to be important in venous thrombosis13, 14. GPIb-IX consists of GPIbα, GPIbβ, GPIX, and GPV15. The extracellular N-terminal region of GPIbα contains the binding sites for VWF and thrombin15, whereas the cytoplasmic domain of GPIbα is linked to the actin cytoskeleton via filamin16, 17. The cytoplasmic domain of GPIb also interacts with 14-3-3ζ18–20. A major binding site for 14-3-3ζ is located in the C-terminal region of GPIbα18, 21. The binding of 14-3-3ζ to the C-terminus of GPIbα is important for regulating the VWF binding function of GPIb-IX and in thrombosis22, 23. In this study, we demonstrate that GPIb-IX plays an important role in the LPS-induced platelet adhesion to inflammatory endothelial cells in vitro, LPS-induced glomerular microvascular thrombosis in vivo, and in the mortality of LPS-challenged mice. We also show that an inhibitor of the ligand binding function of GPIb-IX, based on GPIbα C-terminal sequence, micellar MPαC, prevents LPS-induced thrombosis in microvasculature in glomeruli, ameliorates LPS-induced thrombocytopenia, and decreases the mortality of LPS-challenged mice. Thus, GPIb-IX is a potentially important target for developing new drugs for the management of severe endotoxemia.

Materials and Methods

Materials and methods are detailed in the online supplement. Myristoylated peptides MPαC (C13H27CONH-SIRYSGHpSL) and MCsC (C13H27CONH-LSISYGSHR) were formulated as micelles with PEG2000-DSPE (Avanti Polar Lipids Inc. Alabaster, AL), L-α-phosphatidylcholine (egg PC, Type XI-E, Sigma-Aldrich, St. Louis, MO) and peptides at a molar ratio of 45:5:1(in some experiments, 45:5:2) as previously described24. The IL-4R/Ibα mice lack the gene for endogenous mouse GPIbα but instead express a fusion protein where the extracellular sequences of human GPIbα have been replaced by an extracellular domain from the interleukin-4 receptor25. Platelet adhesion to endothelial monolayers was assayed under shear stress introduced using a cone-plate rheometer (Rheostress 1, Thermo-HAAKE, Paramus, NY)26, 27.

Results

The importance of GPIb-IX in LPS-induced mortality

We have used two approaches: a genetic approach and a selective inhibitor of GPIb-IX function to determine the importance of GPIb-IX in endotoxemia, and the potential of inhibition of GPIb-IX in management of endotoxemia. GPIb-IX deficient mice have significant thrombocytopenia and abnormally sized platelets, and thus cannot be used to dissect the consequence of defects in GPIb-IX function. Thus, we tested LPS-induced mortality in transgenic mice expressing a fusion protein of the IL-4 receptor extracellular domain and GPIbα transmembrane and cytoplasmic domain (IL-4R-Ibα). IL-4R-Ibα lacks the receptor function of GPIb-IX, but preserves the function of the cytoplasmic domain of GPIb to interact with the membrane skeleton, thus maintaining the platelet shape and ameliorating thrombocytopenia in GPIbα−/− mice25, 28. Compared with wild type mice, IL4-Ibα mice showed a significant reduced mortality when challenged with LPS, indicating that a functional GPIb-IX complex on the platelet surface is important in LPS-induced mortality in this mouse endotoxemia model (Fig. 1).

Figure 1. The importance of platelet GPIb-IX in LPS-induced mortality in a mouse endotoxemia model.

Figure 1

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The wild type (WT, n=17) and IL-4R/Ibα (n=16) mice were challenged by intraperitoneal injection of LPS (100 µg) and their mortality monitored. Mouse survival rate was estimated using Kaplan-Meier plot. Logrank test shows that the difference between WT and IL-4R/Ibα is highly significant (p<0.002).

Micellar MPαC and its effects on GPIb-IX-dependent platelet function

To determine the role of GPIb-IX in human and mouse platelet adhesion and in vivo thrombosis during endotoxemia and to evaluate the potential of targeting GPIb, it is necessary to develop GPIb-IX inhibitors. However, a common problem in developing GPIb-IX inhibitors is that the GPIb extracellular domain-binding molecules and antibodies induce severe thrombocytopenia in vivo. To overcome this problem, we applied knowledge of the important role of the GPIbα cytoplasmic domain 14-3-3 binding site in GPIb-IX function, and developed a phosphotidylcholine/DSPE-PEG2000 micellar delivery system incorporating a myristoylated peptide, Myr-SIRYSGHpSL (MPαC), corresponding to the C-terminal 14-3-3ζ binding sequence of GPIbα, and a myristoylated scrambled control peptide (MCsC). The micellar formulation allows safe in vivo use of the highly hydrophobic MPαC without requiring toxic solvents. Indeed, no noticeable adverse effect was observed in mice following retro-orbital injection of micellar MPαC and control micellar MCsC, which have similar pharmacokinetics in the circulation (Supplemental Fig. I). To examine whether micellar MPαC inhibited 14-3-3ζ binding to GPIb-IX, micellar MPαC and MCsC were preincubated with platelets. Platelets were then solubilized and immunoprecipitated with an anti-GPIbα antibody. Micellar MPαC, but not micellar MCsC, inhibited the co-immunoprecipitation of GPIb-IX and 14-3-3ζ by ~80% (Fig. 2A, 2B), indicating that micellar MPαC is effective in blocking 14-3-3ζ binding to GPIb-IX.

Figure 2. Selective inhibition of GPIb-IX-dependent platelet function and arterial thrombosis by micellar MPαC.

Figure 2

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(A) Platelets were preincubated with MPαC or control MCsC, solubilized and then immunoprecipitated with an anti-GPIbα antibody (LJ-P3). Immunoprecipitates were immunoblotted with anti-GPIbα and anti-14-3-3ζ antibodies. (B) GPIb-bound 14-3-3ζ was quantified by measuring immunoblot band intensity (mean ± SD, 3 experiments, t-test). (C) Human platelet-rich plasma (PRP) was preincubated with or without increasing concentrations of the MPαC or control peptide for 15 minutes, and stimulated with ristocetin (1.2 mg/ml) to induce platelet aggregation. (D) Human PRP was preincubated with 12.5 µM MPαC or MCsC for 15 minutes and stimulated with ADP (5 µM), collagen (2 µg/ml) and the thromboxane A2 analog, U46619 (1.6 µM) (shown in the figure are representative aggregation curves from 3 independent experiments using blood from 3 donors). (E) C57BL/6J mice were retro-orbitally injected with MPαC or control peptide (2 µmol/kg) 15 minutes before the carotid artery injury by ferric chloride (10%). Blood flow was recorded until vessel occlusion. The experiment was repeated 10 times using 10 mice for each group (MPαC and MCsC, P< 0.01, t-test with Welch correction). Each triangle represents the occlusion time of each mouse.

To determine whether micellar MPαC affects GPIb-dependent platelet function, human platelet rich-plasma was pre-treated with micellar MPαC or control micellar MCsC. VWF/GPIb-IX-dependent platelet agglutination/aggregation was induced by adding ristocetin, which allows soluble VWF binding to GPIb-IX. Micellar MPαC dose-dependently inhibited ristocetin-induced human platelet aggregation (Fig. 2C). In contrast to the inhibitory effect of MPαC on VWF/GPIb-IX-mediated platelet aggregation, micellar MPαC treatment of platelet-rich plasma had no significant effect on platelet aggregation induced by the GPIb-IX-independent agonists ADP, collagen, and U46619 (a thromboxane A2 analogue) (Fig. 2D). Thus, micellar MPαC selectively inhibits GPIb-IX-dependent platelet function without inhibiting general platelet activation signaling.

The effect of micellar MPαC on arterial thrombosis in vivo

GPIb-IX is known to play an important role in arterial thrombosis29. Thus, we evaluated the in vivo effect of micellar MPαC on arterial thrombosis using the ferric chloride (FeCl3)-induced mouse carotid artery thrombosis model. Retro-orbital injection of micellar MPαC significantly (P<0.01) delayed FeCl3-induced carotid artery occlusive thrombosis compared to the control (Fig. 2E), indicating that micellar MPαC is an effective inhibitor of arterial thrombosis in vivo.

LPS-induced GPIb-IX-dependent platelet adhesion to vascular endothelial cells and the inhibitory effect of MPαC

Although LPS is known to promote injury-induced thrombosis 5, 30, it is unclear how LPS alone induces thrombosis in endotoxemia. The role of platelets in thrombosis under high shear rate flow conditions allows us to hypothesize that GPIb-IX-dependent platelet adhesion to the vascular endothelium may play a role in initiating LPS-induced microvascular thrombosis. To test this hypothesis, we investigated whether LPS can directly induce platelet adhesion to vascular endothelium under high shear rate conditions. A confluent monolayer of human umbilical vein endothelial cells (HUVECs) was pretreated with LPS before exposure to washed human platelets under flow at 800 s−1 shear rate. As expected, platelets poorly adhered to unstimulated endothelial cell surfaces (Fig.3, A, B, and C). LPS treatment of endothelial cells, however, slowly and transiently induced platelet adhesion to HUVEC cells, with a maximal effect at 1 hour (Fig. 3A). Treatment of platelets with micellar MPαC, but not the control micellar MCsC, inhibited LPS-induced platelet adhesion (Fig. 3, B and C). Similarly, an anti-GPIbα monoclonal antibody, LJ-P3, also inhibited platelet adhesion to LPS-stimulated HUVEC cells (Fig. 3D). Importantly, the presence of isolated human leukocytes greatly enhanced and accelerated LPS-induced platelet adhesion to endothelial cells, which was also inhibited by MPαC (Fig. 3E). These data suggest that LPS induces platelet adhesion to the vascular endothelium by leukocyte-dependent and independent mechanisms. These data also suggest that LPS-induced platelet adhesion to endothelial cells requires GPIb-IX function, and that MPαC antagonizes LPS-induced platelet adhesion to the vascular endothelium.

Figure 3. The effect of MPαC and an anti-GPIb antibody on platelet adhesion to LPS stimulated endothelial cells under shear stress.

Figure 3

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(A) HUVEC monolayers were preincubated without (0 hour) or with LPS (20 µM) for various lengths of time. Washed platelets (3X108/ml) were then allowed to adhere to these HUVECs under constant shear rate (800s−1) for 5 minutes. (B) HUVEC monolayers were stimulated without or with LPS for 1 hour. Platelets (3X108/ml) were preincubated with micellar peptides (50 µM) for 30 minutes, and then allowed to adhere to HUVECs under a constant shear rate (800s−1) for 5 minutes. (C) Representative pictures from (B) Size of the scale bar = 25 µm. (D) Platelets were preincubated without or with 10 µg/ml of an inhibitory anti-GPIbα antibody LJ-P3 or IgG control for 30 minutes, and then allowed to adhere to LPS-stimulated or control HUVEC monolayers as in (B). (E) HUVECs were preincubated with or without LPS for 5 minutes. Platelets were preincubated with or without 50 µM MPαC or MCsC and then allowed to adhere to HUVECs under shear stress for 5 minutes with or without simultaneous addition of peripheral blood leukocytes. Platelets directly adhering to the HUVEC monolayer in each assay were quantified (mean ± SD, 3 experiments). Statistic significance was determined using nonparametric ANOVA in all assays in this figure.

The effect of MPαC in LPS-induced microvascular thrombosis in an endotoxemia mouse model

To investigate whether MPαC attenuates LPS-induced microvascular thrombosis, we examined the effect of micellar MPαC on LPS-induced microvascular thrombosis in the kidney glomeruli of C57/BL mice. Intraperitoneal injection of LPS induced microvascular thrombosis in kidney glomeruli, as indicated by Mallory’s phosphotungstic acid hematoxylin staining of platelets and fibrin deposition (Fig. 4A). LPS-induced microvascular thrombosis was significantly reduced in micellar MPαC-treated mice, as compared with the MCsC micelles (Fig 4A). Similar results were also obtained with an immunohistochemical staining of microvascular thrombi with an anti-platelet integrin αIIb antibody (Fig. 4B). Thus, our data indicate that GPIb-IX plays a critical role in LPS-induced microvascular thrombosis in vivo, and that disruption of GPIb-dependent platelet function by MPαC is effective in preventing LPS-induced microvascular thrombosis.

Figure 4. The effect of MPαC on LPS induced microvascular thrombosis in kidney glomeruli.

Figure 4

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(A) C57BL mice were retro-orbitally injected with 2 µmol/kg MPαC or control MCsC prior to intraperitoneal injection of LPS (22 mg/kg). Kidney sections from peptide treated- or control mice were stained with phosphotungstic acid hematoxylin to show fibrin and platelets in the glomeruli. The experiment was repeated 6 times using 6 different mice for each group. Bars indicate the average (±SD) of the percentage of phosphotungstic acid hematoxylin stained area per glomerulus (50 random glomeruli from 6 mice/group) (*P<0.001, t-test with Welch correction). (B) Kidney sections from MPαC- or MCsC-treated mice or control mice were also immunostained with an anti-integrin αIIb monoclonal antibody to show the platelet-rich thrombi. Shown in the figure is a typical image from 6 experiments (6 mice). Arrows point to glomeruli in which microvascular thrombi are concentrated.

The effect of micellar MPαC and Integrilin on survival rate in LPS-challenged mice

The effect of MPαC in alleviating LPS-induced platelet-endothelial cell adhesion and microvascular thrombosis suggests the potential to use MPαC in the management of endotoxemia. To determine the effect of MPαC on endotoxemic mice, we assessed the mortality in mice challenged with LPS. Injection of a high concentration of LPS (22 mg/kg) caused 70% mortality in control mice within 48 hours (Fig. 5A). Injection of the control micellar MCsC had no significant effect on LPS-induced mortality (Fig. 5A). In comparison, a single dosage of micellar MPαC injection before LPS challenge significantly reduced LPS-induced mortality to ~30% (P=0.02) (Fig. 5A), suggesting the important role of GPIb-IX-dependent platelet adhesion in the process. Interestingly, injection of MPαC 1 hour after LPS challenge also significantly reduced mortality in comparison with scrambled control MCsC, suggesting a possible therapeutic potential (Fig. 5B). Consistent with the importance of platelets in LPS-induced mortality, injection of an integrin inhibitor, Integrilin, similarly showed a trend in reducing the LPS-induced mortality (Fig. 5C), indicating a role for the platelet-dependent microvascular thrombosis in the grave consequences of endotoxemia, and the beneficial effect of anti-platelet therapy. While the effects of MPαC and Integrilin in reducing mortality are similar, injection of micellar MPαC to mice only mildly prolonged the tail bleeding time (Fig. 5D). In contrast, Integrilin caused dramatically prolonged tail bleeding time in mice (Fig. 5E). These findings indicate that GPIb-IX-dependent microvascular thrombosis contributes to mortality in sepsis. Our results also suggest that micellar MPαC has the potential to be developed into an effective agent in reducing LPS-induced mortality with only a very mild tendency towards bleeding side effect.

Figure 5. The effect of MPαC on the survival probability of LPS-challenged mice.

Figure 5

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(A) C57BL/6 mice retro-orbitally injected with micellar MPαC (2 µmol/kg) (n=20) or MCsC (n=20), and mice without micellar peptide injection (No treatment) were intraperitoneally injected with LPS (22 mg/kg). Mouse survival rate was estimated using the Kaplan-Meier plot (20 mice/group, p=0.02, MPαC vs MCsC). (B). C57BL/6 mice were first injected with LPS, one hour prior to injection of MPαC (10µmol/kg) (n=10) or MCsC control (n=10) micelles. Survival rates at different time points were recorded as in (A). (C) C57BL/6 mice were retro-orbitally injected with or without Integrilin (5 mg/kg), prior to intraperitoneal LPS injection. Mouse survival rate was estimated using the Kaplan-Meier plot (10 mice/group). (D) C57BL/6 mice were retro-orbitally injected with micellar 2 µmol/kg MPαC or control MCsC 15 minutes before transection of the tail. The time to the cessation of tail bleeding was recorded and shown as closed triangles. The experiment was repeated using multiple mice as indicated for each group (MCsC, n=16, MPαC, n=17, no treatment n=22, P=0.23, Kruskal-Wallis non-parametric ANOVA, median are shown as bars). (E) C57BL/6 mice were retro-orbitally injected with Integrilin (Integrilin,5 mg/kg) (n=10) or saline (n=10) 5 minutes before tail bleeding time analysis (*P<0.01, Mann-Whitney test, median value are indicated as bars).

The effect of micellar MPαC on LPS-induced thrombocytopenia

It is known that severe sepsis in patients or injection of LPS in mice is associated with thrombocytopenia. Thus, we investigated whether MPαC or Integrilin affected LPS-induced changes in circulating platelet counts. Intraperitoneal injection of 12 mg/kg LPS caused a platelet count decrease of ~ 50% 4 hours following LPS injection (Fig. 6A). A single injection of micellar MPαC, partially, but significantly mitigated LPS-induced thrombocytopenia (Fig. 6A). In contrast to the significant effect of MPαC, injection of 5 mg/kg Integrilin had no effect on LPS-induced thrombocytopenia (Fig. 6B) despite its significant effect in prolonging bleeding time (Fig. 5E) and in reducing mortality (Fig. 5C). To exclude the possibility that MPαC affected platelet counts independent of LPS, we also examined platelet counts in healthy mice treated with micellar MPαC or the control micellar peptide MCsC. MPαC did not induce changes in platelet counts without LPS injection (Fig. 6C). These data indicate that LPS-induced thrombocytopenia involves GPIb-IX function but not that of integrin αIIbβ3. Thus, LPS-induced thrombocytopenia may not be a direct consequence of thrombus formation, but a consequence of GPIb-IX-dependent platelet clearance. These data also indicate that micellar MPαC is effective in mitigating endotoxemia-induced thrombocytopenia.

Figure 6. The effect of MPαC on LPS-induced thrombocytopenia.

Figure 6

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(A) C57BL/6 mice were retro-orbitally injected with 2 µmol/kg MPαC or control MCsC prior to intraperitoneal (i.p.) injection of LPS (12 mg/kg) (10 mice/group). Blood platelet counts were obtained before and after LPS challenge for 2 and 4 hours. The experiment was repeated 15 times using 15 different mice. The percentage of platelets remaining in the peripheral blood is shown (mean±SD, * P<0.05, n=15/group). (B) Relative platelet counts of C57BL/6 mice retro-orbitally injected with Integrilin (5 mg/kg) or saline before, 2 hours, and 4 hours after challenge with LPS (12 mg/kg) i.p. injection (repeated using 10 mice/group, mean±SD). (C) Platelet counts of C57BL/6 mice before and 4 hours after retro-orbitally injected with MPαC (2 µmol/kg) or vehicle (buffer) only (repeated using 10 mice/group, mean±SD).

Discussion

Our data indicate that GPIb-IX plays an important role in platelet adhesion to LPS-stimulated vascular endothelial cells, microvascular thrombosis and LPS-induced thrombocytopenia and mortality. In addition, we show that a GPIb inhibitor based on the C-terminal sequence of the cytoplasmic domain of GPIbα is effective in ameliorating LPS-induced microvascular thrombosis, thrombocytopenia, and mortality in vivo. These data suggest a potential new strategy for the management of endotoxemia-induced microvascular thrombosis and consequent multiple organ failure, and for improving the survival rate of endotoxemia patients.

It is currently recognized that poor prognosis of endotoxemia in sepsis is associated with systemic microvascular thrombosis and disseminated intravascular coagulation1, 2. However, the mechanism that initiates microvascular thrombosis in endotoxemia remains unclear. The current concept emphasizes the initiation of the coagulation cascade. Thus, the clinical anti-thrombotic treatment of severe sepsis is limited to anti-coagulant agents such as recombinant activated protein C3. Our study suggests that platelet adhesion, particularly GPIb-IX-dependent platelet adhesion, is important in LPS-induced microvascular thrombus formation. Thus, inhibitors of platelet adhesion appear to reduce LPS-induced platelet-endothelial cell interaction, microvascular thrombosis and mortality of LPS-treated mice. These data are consistent with the concept that platelet adhesion, particularly GPIb-IX-dependent platelet adhesion, is important for thrombosis in microcirculation10, and suggest the potential for GPIb-IX inhibitors as anti-thrombotics in the management of endotoxemia.

The importance of platelet GPIb-IX in endotoxemia and LPS-induced microvascular thrombosis is supported by experiments using a combination of genetic manipulation and pharmacological inhibitors in mouse models: A major challenge in studying endotoxemia and sepsis are the animal endotoxemia models may not precisely replicate the wide range of pathophysiologic events occurring in humans. Thus, results obtained with mouse or other animal models, while very valuable as a guide and theoretical basis, need to be carefully evaluated in treating human endotoxemia. A more extreme viewpoint on this issue was published in a recent report proposing bypassing animal models for the reason that the induced gene expression patterns are different between humans and mice under inflammatory conditions31. However, that report did not compare gene expression or consequent functional responses between human and mouse megakaryocytes and platelets in their models. More importantly, the data from that publication are not sufficient to exclude the similarities between humans and mice in inflammatory response. For example, comparison of induced gene expression patterns is unlikely to reveal the importance of specific molecular mechanisms of inflammation that may be similar between humans and mice. Furthermore, although different in overall pattern, there is about 47–61% similarity in gene changes between human and mice in response to inflammatory stimuli as shown in that study31. It is possible that these similarly changed genes may represent those specific molecules that play key roles in inflammatory responses. It is important to note that conducting severe endotoxemia models similar to the stringently controlled animal experiments is unethical to perform in humans. Furthermore, it is well-established that human, mice, and other animals share very similar molecular mechanisms and genetic bases in physiology and pathology including thrombosis and inflammation. Thus, animal models are often the only available means for the medical community to study severe endotoxemia (and many other severe diseases) in order to obtain knowledge regarding the molecular mechanisms and discover drug targets for the treatment of human diseases. This approach has proven valuable. Abandoning of animal models by over-emphasis of the difference between humans and animals will prove detrimental to our progress in understanding and development of new treatment for severe human diseases.

To be able to further test whether GPIb-IX-mediated platelet adhesion and activation is important in severe endotoxemia in humans, it is necessary to develop new drugs that can interfere with GPIb-IX function in vivo in endotoxemic patients. In this regard, MPαC is a new type of inhibitor that selectively interferes with GPIb-IX function without affecting GPIb-IX-independent platelet activation pathways. Unlike the GPIb-IX antagonists currently under development, MPαC acts on the cytoplasmic domain of GPIbα and does not induce thrombocytopenia. In fact, a major novelty of this study is the discovery that MPαC alleviates thrombocytopenia induced by LPS. Micellar MPαC represents not only a potential new strategy for the management of endotoxemia-induced microvascular thrombosis and potentially for improving the survival rate of sepsis patients, but also the only inhibitor that inhibits GPIb-IX-dependent platelet clearance. Interestingly, in hemolytic uremic syndrome, bacterial shiga toxin has been known to induce thrombotic microangiopathy and consumptive thrombocytopenia32. In inherited thrombotic thrombocytopenic purpura, VWF-GPIb interaction also causes microvascular thrombosis and thrombocytopenia. Thus, it would be interesting to further investigate whether MPαC can also be used in the treatment of hemolytic uremic syndrome and other types of thrombotic microangiopathy that involves VWF binding to GPIb-IX. Nevertheless, although we conclude that MPαC exerts its effects mainly through inhibiting 14-3-3 binding to GPIb, we do not totally exclude the possibility of additional effects of MPαC on other functions of 14-3-3. However, such additional effects of MPαC, if present, are also likely to involve the GPIb-IX signaling pathway because MPαC does not significantly affect GPIb-IX-independent platelet activation and mimics the anti-thrombotic effect seen in GPIb-IX functional deficiency.

Our data indicate that endothelial cell exposure to LPS alone is sufficient to induce a slow endothelial cell response that allows GPIb-IX-dependent platelet adhesion. However, LPS rapidly and potently induces platelet adhesion to endothelial cells in the presence of peripheral blood leukocytes, indicating that inflammatory mediators released by LPS-stimulated leukocytes induce rapid exposure of GPIb-IX ligands on endothelial cell surfaces. Large quantities of VWF are stored in endothelial Weibel-Palade bodies and become exposed on the endothelial cell surface upon stimulation by inflammatory mediators33, 34. It has been reported that platelets adhere to inflammatory mediator (histamine)-activated endothelial cells and form a “beads-on-a-string” like structure due to exposure of ultra-large VWF multimers on the endothelial surface34. Thus, it is likely that LPS and inflammatory mediator-induced exposure of endothelial-bound VWF is important in mediating GPIb-IX-dependent platelet adhesion. In this respect, suppression of the VWF-cleaving activity of ADAMTS13 has been reported in patients suffering from sepsis 35. Also, increased VWF levels and the presence of more active ultra-large VWF in plasma have been found in severe sepsis and septic shock patients36. In a sepsis model, VWF-deficient mice lived significantly longer than wild type mice37, although an earlier report by some of the coauthors of that work did not show significant difference between VWF-deficient mice and wild type controls following LPS challenge38. It is also possible that the role of GPIb-IX may be mediated by its binding to endothelial P-selectin 39 and endothelial-bound coagulation factors (such as thrombin)40 or through leukocyte or endothelial integrins41, 42, although there have been studies challenging the role of P-selectin and integrins in platelet string formation on endothelial cells43. It is interesting to further investigate the mechanism by which GPIb-IX mediates microvascular thrombosis in endotoxemia and whether an anti-platelet therapy will have an advantage over anti-VWF therapy in treating platelet-dependent microvascular thrombosis.

Our data not only indicate that MPαC inhibits LPS-induced microvascular thrombosis, but also that it alleviates thrombocytopenia induced by LPS. Consumptive thrombocytopenia in sepsis and endotoxemia is associated with poor prognosis1, 2. Apart from the deposition of activated platelets in microvascular thrombi, thrombocytopenia is mainly caused by the clearance of LPS-stimulated platelets from the circulation by the liver and spleen44, 45. Interestingly, a recent study suggests that clearance of both VWF and platelets involves Ashwell receptors in hepatocytes44, 46. Thus, it is tempting to speculate LPS-induced consumptive thrombocytopenia is partially dependent upon Ashwell-receptor recognition of platelet-bound VWF or Ashwell receptor recognition of a VWF-bound conformational state of GPIb-IX. In this respect, it is worth noting that MPαC is unique as a GPIb-IX inhibitor, because it inhibits GPIb-IX function by competitively blocking 14-3-3 binding to GPIb, thus restricting GPIb-IX to a conformational state that is unable to bind VWF. Clearly, this conformation of GPIb-IX also prevents clearance of platelets from the circulation. In summary, this study shows that GPIb-IX plays an important role in severe endotoxemia, and an inhibitor of GPIb-IX function alleviates LPS-induced platelet adhesion, microvascular thrombosis, and consumptive thrombocytopenia which may potentially be effective in the management of endotoxemia in sepsis.

Supplementary Material

supp1
supp2

Significance.

This work demonstrates the importance of the platelet adhesion receptor, GPIb-IX, in endotoxemia, and the in vivo effects of an inhibitor of GPIb-IX interaction with its intracellular binding partner, 14-3-3 protein, in reducing endotoxemia-induced microvascular thrombosis in glomeruli, thrombocytopenia and mortality. These results provide a new mechanism for the endotoxemia-induced microvascular thrombosis and thrombocytopenia, and suggest potential for the GPIb-IX inhibitor in managing endotoxemia.

Acknowledgements

We thank Dr. William Fay for helping with the FeCl3-induced carotid artery thrombosis model, Dr. Zaverio Ruggeri for providing LJ-P3, and Keegan Delaney, Brian Estevez and Kelly O’Brien for proof reading the manuscript.

Sources of funding

This work was supported by Grants from NHLBI/NIH, HL062350, HL068819 and HL080264 to XD, HL50545 to JW, and AHA Predoctoral Fellowships to HY and AC.

Abbreviations

LPS

lipopolysaccharides

GPIb-IX

the glycoprotein Ib-IX complex

VWF

von Willebrand factor

HUVEC

human umbilical vein endothelial cells.

Footnotes

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Disclosure

X. D. holds a patent relevant to this study. No additional conflict of interests to declare.

References

  • 1.Opal SM, Esmon CT. Bench-to-bedside review: Functional relationships between coagulation and the innate immune response and their respective roles in the pathogenesis of sepsis. Crit Care. 2003;7:23–38. doi: 10.1186/cc1854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Hotchkiss RS, Karl IE. The pathophysiology and treatment of sepsis. N Engl J Med. 2003;348:138–150. doi: 10.1056/NEJMra021333. [DOI] [PubMed] [Google Scholar]
  • 3.Bernard GR, Vincent JL, Laterre PF, LaRosa SP, Dhainaut JF, Lopez-Rodriguez A, Steingrub JS, Garber GE, Helterbrand JD, Ely EW, Fisher CJ., Jr Efficacy and safety of recombinant human activated protein c for severe sepsis. N Engl J Med. 2001;344:699–709. doi: 10.1056/NEJM200103083441001. [DOI] [PubMed] [Google Scholar]
  • 4.Benefield RJ, Drevets DA, Huycke MM, Gentry CA. A multicenter evaluation of the safety of drotrecogin alfa (activated) in patients with baseline bleeding precautions. Current drug safety. 2012;7:3–7. doi: 10.2174/157488612800492735. [DOI] [PubMed] [Google Scholar]
  • 5.Zhang G, Han J, Welch EJ, Ye RD, Voyno-Yasenetskaya TA, Malik AB, Du X, Li Z. Lipopolysaccharide stimulates platelet secretion and potentiates platelet aggregation via tlr4/myd88 and the cgmp-dependent protein kinase pathway. J Immunol. 2009;182:7997–8004. doi: 10.4049/jimmunol.0802884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Shibazaki M, Nakamura M, Endo Y. Biphasic, organ-specific, and strain-specific accumulation of platelets induced in mice by a lipopolysaccharide from escherichia coli and its possible involvement in shock. Infect Immun. 1996;64:5290–5294. doi: 10.1128/iai.64.12.5290-5294.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Shibazaki M, Kawabata Y, Yokochi T, Nishida A, Takada H, Endo Y. Complement-dependent accumulation and degradation of platelets in the lung and liver induced by injection of lipopolysaccharides. Infect Immun. 1999;67:5186–5191. doi: 10.1128/iai.67.10.5186-5191.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Davis RB, Meeker WR, Mc QD. Immediate effects of intravenous endotoxin on serotonin concentrations and blood platelets. Circ Res. 1960;8:234–239. doi: 10.1161/01.res.8.1.234. [DOI] [PubMed] [Google Scholar]
  • 9.Davis RB, Palmer MJ. Thrombocytopenia and release of platelet amines induced by thrombin and bacterial lipopolysaccharide. Observations after infusion of low molecular weight dextran. Br J Exp Pathol. 1965;46:554–563. [PMC free article] [PubMed] [Google Scholar]
  • 10.Ruggeri ZM, Mendolicchio GL. Adhesion mechanisms in platelet function. Circ Res. 2007;100:1673–1685. doi: 10.1161/01.RES.0000267878.97021.ab. [DOI] [PubMed] [Google Scholar]
  • 11.Du X. Signaling and regulation of the platelet glycoprotein ib-ix-v complex. Curr Opin Hematol. 2007;14:262–269. doi: 10.1097/MOH.0b013e3280dce51a. [DOI] [PubMed] [Google Scholar]
  • 12.Li Z, Delaney MK, O'Brien KA, Du X. Signaling during platelet adhesion and activation. Arterioscler Thromb Vasc Biol. 2010;30:2341–2349. doi: 10.1161/ATVBAHA.110.207522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Brill A, Fuchs TA, Chauhan AK, Yang JJ, De Meyer SF, Kollnberger M, Wakefield TW, Lammle B, Massberg S, Wagner DD. Von willebrand factor-mediated platelet adhesion is critical for deep vein thrombosis in mouse models. Blood. 2011;117:1400–1407. doi: 10.1182/blood-2010-05-287623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Joglekar MV, Ware J, Xu J, Fitzgerald ME, Gartner TK. Platelets, glycoprotein ib-ix, and von willebrand factor are required for fecl(3)-induced occlusive thrombus formation in the inferior vena cava of mice. Platelets. 2012 doi: 10.3109/09537104.2012.696746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Ware J. Molecular analyses of the platelet glycoprotein ib-ix-v receptor. Thromb Haemost. 1998;79:466–478. [PubMed] [Google Scholar]
  • 16.Fox JE. Identification of actin-binding protein as the protein linking the membrane skeleton to glycoproteins on platelet plasma membranes. J Biol Chem. 1985;260:11970–11977. [PubMed] [Google Scholar]
  • 17.Andrews RK, Fox JE. Identification of a region in the cytoplasmic domain of the platelet membrane glycoprotein ib-ix complex that binds to purified actin-binding protein. J Biol Chem. 1992;267:18605–18611. [PubMed] [Google Scholar]
  • 18.Du X, Fox JE, Pei S. Identifcation of a binding sequence for the 14-3-3 protein within the cytoplasmic domain of the adhesion receptor, platelet glycoprotein ibα. J Biol Chem. 1996;271:7362–7367. doi: 10.1074/jbc.271.13.7362. [DOI] [PubMed] [Google Scholar]
  • 19.Andrews RK, Harris SJ, McNally T, Berndt MC. Binding of purified 14-3-3 zeta signaling protein to discrete amino acid sequences within the cytoplasmic domain of the platelet membrane glycoprotein ib-ix-v complex. Biochemistry. 1998;37:638–647. doi: 10.1021/bi970893g. [DOI] [PubMed] [Google Scholar]
  • 20.Mangin P, David T, Lavaud V, Cranmer SL, Pikovski I, Jackson SP, Berndt MC, Cazenave JP, Gachet C, Lanza F. Identification of a novel 14-3-3{zeta} binding site within the cytoplasmic tail of platelet glycoprotein ib{alpha} Blood. 2004;104:420–427. doi: 10.1182/blood-2003-08-2881. [DOI] [PubMed] [Google Scholar]
  • 21.Bodnar RJ, Gu M, Li Z, Englund GD, Du X. The cytoplasmic domain of the platelet glycoprotein ibalpha is phosphorylated at serine 609. J Biol Chem. 1999;274:33474–33479. doi: 10.1074/jbc.274.47.33474. [DOI] [PubMed] [Google Scholar]
  • 22.Dai K, Bodnar R, Berndt MC, Du X. A critical role for 14-3-3zeta protein in regulating the vwf binding function of platelet glycoprotein ib-ix and its therapeutic implications. Blood. 2005;106:1975–1981. doi: 10.1182/blood-2005-01-0440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Jain S, Zuka M, Liu J, Russell S, Dent J, Guerrero JA, Forsyth J, Maruszak B, Gartner TK, Felding-Habermann B, Ware J. Platelet glycoprotein ib alpha supports experimental lung metastasis. Proc Natl Acad Sci U S A. 2007;104:9024–9028. doi: 10.1073/pnas.0700625104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Krishnadas A, Rubinstein I, Onyuksel H. Sterically stabilized phospholipid mixed micelles: In vitro evaluation as a novel carrier for water-insoluble drugs. Pharm Res. 2003;20:297–302. doi: 10.1023/a:1022243709003. [DOI] [PubMed] [Google Scholar]
  • 25.Kanaji T, Russell S, Ware J. Amelioration of the macrothrombocytopenia associated with the murine bernard-soulier syndrome. Blood. 2002;100:2102–2107. doi: 10.1182/blood-2002-03-0997. [DOI] [PubMed] [Google Scholar]
  • 26.Yin H, Stojanovic A, Hay N, Du X. The role of akt in the signaling pathway of the glycoprotein ib-ix induced platelet activation. Blood. 2008;111:658–665. doi: 10.1182/blood-2007-04-085514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Yin H, Liu J, Li Z, Berndt MC, Lowell CA, Du X. Src family tyrosine kinase lyn mediates vwf/gpib-ix-induced platelet activation via the cgmp signaling pathway. Blood. 2008;112:1139–1146. doi: 10.1182/blood-2008-02-140970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Ware J, Russell S, Ruggeri ZM. Generation and rescue of a murine model of platelet dysfunction: The bernard-soulier syndrome. Proc Natl Acad Sci U S A. 2000;97:2803–2808. doi: 10.1073/pnas.050582097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Bergmeier W, Piffath CL, Goerge T, Cifuni SM, Ruggeri ZM, Ware J, Wagner DD. The role of platelet adhesion receptor gpibalpha far exceeds that of its main ligand, von willebrand factor, in arterial thrombosis. Proc Natl Acad Sci U S A. 2006;103:16900–16905. doi: 10.1073/pnas.0608207103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Stark RJ, Aghakasiri N, Rumbaut RE. Platelet-derived toll-like receptor 4 (tlr-4) is sufficient to promote microvascular thrombosis in endotoxemia. PloS one. 2012;7:e41254. doi: 10.1371/journal.pone.0041254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Seok J, Warren HS, Cuenca AG, et al. Genomic responses in mouse models poorly mimic human inflammatory diseases. Proc Natl Acad Sci U S A. 2013;110:3507–3512. doi: 10.1073/pnas.1222878110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Zheng XL, Sadler JE. Pathogenesis of thrombotic microangiopathies. Annu Rev Pathol. 2008;3:249–277. doi: 10.1146/annurev.pathmechdis.3.121806.154311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Andre P, Denis CV, Ware J, Saffaripour S, Hynes RO, Ruggeri ZM, Wagner DD. Platelets adhere to and translocate on von willebrand factor presented by endothelium in stimulated veins. Blood. 2000;96:3322–3328. [PubMed] [Google Scholar]
  • 34.Padilla A, Moake JL, Bernardo A, Ball C, Wang Y, Arya M, Nolasco L, Turner N, Berndt MC, Anvari B, Lopez JA, Dong JF. P-selectin anchors newly released ultralarge von willebrand factor multimers to the endothelial cell surface. Blood. 2004;103:2150–2156. doi: 10.1182/blood-2003-08-2956. [DOI] [PubMed] [Google Scholar]
  • 35.Nguyen TC, Liu A, Liu L, Ball C, Choi H, May WS, Aboulfatova K, Bergeron AL, Dong JF. Acquired adamts-13 deficiency in pediatric patients with severe sepsis. Haematologica. 2007;92:121–124. doi: 10.3324/haematol.10262. [DOI] [PubMed] [Google Scholar]
  • 36.Bockmeyer CL, Claus RA, Budde U, Kentouche K, Schneppenheim R, Losche W, Reinhart K, Brunkhorst FM. Inflammation-associated adamts13 deficiency promotes formation of ultra-large von willebrand factor. Haematologica. 2008;93:137–140. doi: 10.3324/haematol.11677. [DOI] [PubMed] [Google Scholar]
  • 37.Lerolle N, Dunois-Larde C, Badirou I, Motto DG, Hill G, Bruneval P, Diehl JL, Denis CV, Baruch D. Von willebrand factor is a major determinant of adamts-13 decrease during mouse sepsis induced by cecum ligation and puncture. Journal of thrombosis and haemostasis : JTH. 2009;7:843–850. doi: 10.1111/j.1538-7836.2009.03313.x. [DOI] [PubMed] [Google Scholar]
  • 38.Chauhan AK, Walsh MT, Zhu G, Ginsburg D, Wagner DD, Motto DG. The combined roles of adamts13 and vwf in murine models of ttp, endotoxemia, and thrombosis. Blood. 2008;111:3452–3457. doi: 10.1182/blood-2007-08-108571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Romo GM, Dong JF, Schade AJ, Gardiner EE, Kansas GS, Li CQ, McIntire LV, Berndt MC, Lopez JA. The glycoprotein ib-ix-v complex is a platelet counterreceptor for p-selectin. J Exp Med. 1999;190:803–814. doi: 10.1084/jem.190.6.803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Weeterings C, de Groot PG, Adelmeijer J, Lisman T. The glycoprotein ib-ix-v complex contributes to tissue factor-independent thrombin generation by recombinant factor viia on the activated platelet surface. Blood. 2008;112:3227–3233. doi: 10.1182/blood-2008-02-139113. [DOI] [PubMed] [Google Scholar]
  • 41.Simon DI, Chen Z, Xu H, Li CQ, Dong J, McIntire LV, Ballantyne CM, Zhang L, Furman MI, Berndt MC, Lopez JA. Platelet glycoprotein ibalpha is a counterreceptor for the leukocyte integrin mac-1 (cd11b/cd18) J Exp Med. 2000;192:193–204. doi: 10.1084/jem.192.2.193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Wang Y, Sakuma M, Chen Z, Ustinov V, Shi C, Croce K, Zago AC, Lopez J, Andre P, Plow E, Simon DI. Leukocyte engagement of platelet glycoprotein ibalpha via the integrin mac-1 is critical for the biological response to vascular injury. Circulation. 2005;112:2993–3000. doi: 10.1161/CIRCULATIONAHA.105.571315. [DOI] [PubMed] [Google Scholar]
  • 43.Chauhan AK, Goerge T, Schneider SW, Wagner DD. Formation of platelet strings and microthrombi in the presence of adamts-13 inhibitor does not require p-selectin or beta3 integrin. Journal of thrombosis and haemostasis : JTH. 2007;5:583–589. doi: 10.1111/j.1538-7836.2007.02361.x. [DOI] [PubMed] [Google Scholar]
  • 44.Grewal PK, Uchiyama S, Ditto D, Varki N, Le DT, Nizet V, Marth JD. The ashwell receptor mitigates the lethal coagulopathy of sepsis. Nat Med. 2008;14:648–655. doi: 10.1038/nm1760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Hoffmeister KM, Felbinger TW, Falet H, Denis CV, Bergmeier W, Mayadas TN, von Andrian UH, Wagner DD, Stossel TP, Hartwig JH. The clearance mechanism of chilled blood platelets. Cell. 2003;112:87–97. doi: 10.1016/s0092-8674(02)01253-9. [DOI] [PubMed] [Google Scholar]
  • 46.Rumjantseva V, Grewal PK, Wandall HH, Josefsson EC, Sorensen AL, Larson G, Marth JD, Hartwig JH, Hoffmeister KM. Dual roles for hepatic lectin receptors in the clearance of chilled platelets. Nat Med. 2009;15:1273–1280. doi: 10.1038/nm.2030. [DOI] [PMC free article] [PubMed] [Google Scholar]

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Supplementary Materials

supp1
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supp2
NIHMS574833-supplement-supp2.pdf (119KB, pdf)

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