Platelet glycoprotein Ib-IX as a regulator of systemic inflammation

Abstract

Objective: The platelet glycoprotein Ib-IX (GP Ib-IX) receptor is a well-characterized adhesion receptor supporting hemostasis and thrombosis via interactions with von Willebrand factor. We examine the GP Ib-IX/von Willebrand factor axis in murine polymicrobial sepsis, as modeled by cecal ligation and puncture (CLP).

Approach and results: Genetic absence of the GP Ib-IX ligand, von Willebrand factor, prolongs survival after CLP, but absence of the receptor, GP Ib-IX, does not. Because absence of either von Willebrand factor or GP Ib-IX significantly impairs hemostasis and thrombosis, we sought to define additional GP Ib-IX-dependent pathways impacting survival in the CLP model. We document that the absence of GP Ib-IX leads to reduced platelet-neutrophil and platelet-monocyte interactions. Twenty-four hours after CLP, absence of GP Ib-IX coincides with an alteration in cytokine levels, such as tumor necrosis factor-α secreted by monocytes, and increased macrophage-1 antigen expression by neutrophils.

Conclusions: In contrast to the well-characterized proinflammatory properties of platelets, we describe in the CLP model an anti-inflammatory property associated with platelet GP Ib-IX. Thus, a single platelet receptor displays a dual modulatory role in both the thrombotic and inflammatory pathways associated with polymicrobial sepsis. In sharing leucine-rich motifs with toll-like receptors, platelet GP Ib-IX can be considered a multifunctional participant in hemostasis, thrombosis, and the inflammatory cascade. The results highlight a dynamic role for platelets in systemic inflammation and add to the complex pathophysiologic events that occur during the dysregulated coagulation and inflammation associated with sepsis.

Keywords: cytokines; monocytes; neutrophils; platelet glycoprotein GPIb-IX complex; sepsis.

Figure 1. Kaplan-Meier survival curves following CLP

Severe sepsis induced by CLP generated varying mortalities observed over a 5 day period. All wild-types succumbed to septic burden prior to the 72 hour mark. Deletion of the gene encoding VWF (VWF KO) significantly alleviated septic burden as the rate of mortality was reduced with several mice surviving past 5 days (p = 0.003). Conversely, ablation of a functional platelet GP Ib-IX axis (hIL-4R/Ibα) resulted in no discernible differences in survival when compared to wild-type (p = 0.614). N = 8 for each group. P values were determined by a Logrank analysis.

Figure 2. Platelet-neutrophil/monocyte interactions

Whole blood was collected from wild-type, hIL-4R/Ibα and VWF KO mice under static conditions. (A) Representative flow analysis of samples identifying the neutrophil population (Gr-1⁺/CD115⁻) within wild-type, hIL-4R/Ibα or VWF KO blood is shown. Gating exclusively onto a Gr-1⁺/CD115⁻ population (upper left gray box in panel A) allowed for a second gating of associated CD41⁺ (platelet) events within the neutrophil gate. (B) The CD41⁺/Gr-1⁺/CD115⁻ population analyzed as a percent of total Gr-1⁺/CD115⁻ events illustrates platelet-neutrophil interaction based on GP Ib-IX expression. Wild-type and VWF KO express endogenous GP Ib-IX and are indiscernible from one another (p = 0.54) but both exhibit significantly higher platelet-neutrophil levels compared to hIL-4R/Ibα (p < 0.001). This establishes a GP Ib-IX role in augmenting platelet binding to neutrophils. (C) Utilizing CD115⁺ fluorescence to gate exclusively onto monocytes (lower right gray box in panel A) the GP Ib-IX dependent association with platelets is presented. Comparison of the sample groups showed the wild-type and VWF KO strains to be indistinguishable (p = 0.24). Blood from hIL-4R/Ibα mice had significantly lower platelet-monocyte associations relative to wild-type (p = 0.003). This indicates a significant GPIb-IX dependent modulation of platelet binding to monocytes in the circulation. N = 6 for each group.

Figure 3. Platelet/neutrophil and platelet/monocyte interactions following CLP

(A) Twenty four hours after CLP, whole blood from wild-type and hIL-4R/Ibα mice was analyzed via flow cytometry to determine GP Ib-IX mediated platelet-neutrophil or platelet/monocyte events. (B) The percentage of neutrophils bound by platelets is reduced following CLP in both animal strains relative to static conditions. (C) A similar trend was observed in the monocyte gate with an overall reduction in platelet associated events following CLP. Despite the overall reduction in of platelet associated events in both gates, wild type samples did retain elevated interactions with platelets for both the neutrophil (p = 0.04) and monocyte (p = 0.05) populations. N = 6 (Normal) N = 11 (24 hrs post-CLP).

Figure 4. TNFα levels following CLP

(A) Serum was collected from mice under basal conditions as well as 4, 12 and 24 hours post CLP. Under non-septic (normal) conditions, TNFα is undetectable in serum. At 4 and 12 hours wild type and hIL-4R/Ibα samples exhibit indistinguishable serum TNFα levels. A marked difference is observed at 24 hours following sepsis induction. (B) TNFα levels of individual mice 24 hours post-CLP show on average significantly elevated TNFα levels in those mice lacking GP Ib-IX expression (p = 0.005). Comparison of wild-type and VWF KO show no distinction between serum TNFαlevels (p = 0.933). A horizontal bar represents the overall mean. N = 19 (WT); N = 19 (hIL-4R/Ibα); N = 11 (VWF KO).

Figure 5. Inflammatory cytokine and chemokine levels 24 hours post-CLP

Serum levels of 25 different inflammatory mediators were assessed following CLP surgery. Serum collected from mice was analyzed on an individual basis. A total of six different analytes (including TNFα, not shown) secreted primarily by cells of monocyte/macrophage lineage had significantly different concentrations when comparing wild-type and hIL-4R/Ibα serum. Four of the five analytes showed elevated levels in the hIL-4R/Ibα samples; MIP-1β (p = 0.0078), MCP-1 (p = 0.0129), KC (p = 0.0054) and IL-6 (p = 0.0085). Serum levels of IL-15 was reversed with the wild-type strain exhibiting higher serum levels (p = 0.0006). A horizontal bar indicates overall mean. N = 10 for each group.

Figure 6. Mac-1 expression changes 24 hours post-CLP

Blood isolated from wild-type (WT) and hIL-4R/Ibα mice under normal conditions or following CLP was stained using Gr-1 (neutrophils), CD115 (monocytes) and CD11b (Mac-1). Expression of Mac-1 for both the (A) neutrophil and (B) monocyte populations did not differ between strains in the absence of CLP (normal). Twenty four hrs following CLP, hIL-4R/Ibα display increased surface Mac-1 staining on the surface of neutrophils (p = 0.0348). Monocyte Mac-1 did not differ between strains 24 hours post-CLP. N = 6 for Normal samples and N = 11 for CLP samples.