Biochemical and biomechanical factors regulating leukocyte-thrombus interactions

Background: Whilst the central role of platelets in haemostasis and thrombosis has long been recognised, there is a growing body of evidence supporting an important proinflammatory function for platelets, linked to host defence and a variety of autoimmune and inflammatory diseases. Additionally, α-thrombin the final effector protease of blood coagulation, plays a central role in regulating thrombo-inflammation via broad physiologic functions including coagulation and fibrin generation as well as activation of platelets and other cell types through cleavage of protease-activated receptors (PARs). However, despite its clinical importance, the mechanisms by which microvascular thrombi and generation of α-thrombin regulate leukocyte recruitment to sites of vascular injury remains poorly understood. Aim: To investigate the biochemical and biomechanical factors regulating leukocyte-thrombus interactions at sites of vascular injury. Key findings: Complementary studies utilising an intestinal ischaemia-reperfusion (I/R) injury model as well as a mechanical endothelial injury model in the mesenteric circulation of mice demonstrated that both models induce endothelial cell injury with consequent phosphatidylserine (PS) exposure. Strikingly these PS positive endothelial cells serve as “hot spots” for platelet thrombus formation associated with efficient leukocyte recruitment. Studies utilising particle image velocimetry and computational fluid dynamics revealed that rheological factors significantly impacted leukocyte-thrombus interactions. Thrombi induced local shear alterations and low shear pockets around the base of thrombi where leukocyte recruitment preferentially occurred. Furthermore, the prevailing bulk flow rate was an additional significant modulator of leukocyte-thrombus interactions. As the 3-dimensional growth of thrombi represents a physical barrier to leukocyte migration, a surprising finding of these studies was the ability of leukocytes to migrate through thrombi towards the site of vascular injury. This phenomenon was contingent upon α-thrombin generation. Studies utilising a combination of pharmacologic α-thrombin inhibition and PAR4-/- mice demonstrated that thrombin-dependent cleavage of PAR4 is a key event promoting leukocyte migration through thrombi, via the generation of a platelet CXCR1/2 agonist (predominantly neutrophil-activating peptide-2 [NAP-2]) chemokine gradient. The centrality of NAP-2 in this phenomenon was confirmed through both pharmacologic inhibition and generation of a NAP-2-/- mouse. Interestingly, leukocyte-thrombus interactions were independent of fibrin generation, which in fact negatively regulated leukocyte migration through thrombi. In contrast, cleavage of fibrin by recombinant tissue plasminogen activator or inhibition of fibrin generation enhanced leukocyte migration through thrombi towards sites of endothelial injury. Fibrin(ogen) and platelet GPIbα are major binding sites for α-thrombin. In a murine model of abrogated GPIbα-thrombin binding, both of these interactions negatively regulated leukocyte-thrombus interactions, likely through sequestration of catalytically active α-thrombin. Conclusion: These studies identify the key biochemical factors [α-thrombin generation, platelet derived CXCR1/2 chemokines, platelet P-selectin expression and fibrin(ogen)/GPIbα-thrombin binding] and biomechanical factors [physical-barrier imposed by fibrin, loco-regional rheological effects imparted by thrombus size and prevailing bulk flow] that act in concert to regulate leukocyte recruitment by microvascular thrombi. A number of these factors represent potential therapeutic targets for the modulation of thrombo-inflammatory responses