P-selectin blockade effectively prevents trauma-induced microvascular obstruction

Abstract Background: Trauma is a leading cause of morbidity and mortality worldwide. Although many patients survive the initial blood loss, they are at high risk of developing the multiple organ dysfunction syndrome (MODS). Severely injured individuals often show signs of impaired microvascular perfusion and vascular obstruction, even in organs distal to the site of trauma. These abnormalities can persist despite resuscitation efforts that restore normal arterial blood pressure. Persistent microcirculatory obstruction is directly linked to the development of organ failure and increased mortality. However, it is still not known when after trauma this process begins or what are its underlying mechanisms. Aim: To investigate the cellular and molecular mechanisms underlying microvascular obstruction following trauma using a mouse model of polytrauma, and to determine whether anti-P-selectin treatment improves trauma-induced microvascular perfusion. Methods: The impact of trauma on microvascular circulation in mice was studied by intravital microscopy. Male C57BL/6J WT mice were anesthetized, the jugular vein was cannulated, and the cremaster microcirculation was surgically exposed for microscopic monitoring. Fluorescently conjugated antibodies specific to platelets, leukocytes, and fibrin were injected intravenously, and the cremaster microcirculation at baseline was recorded. Mice were then subjected to a standard polytrauma protocol that included liver, bowel, and quadriceps muscle crush injuries and femur fracture. Immediately after the polytrauma, platelet-leukocyte-endothelial interactions in the cremaster microcirculation were monitored microscopically for up to 30 min. The effect of P-selectin inhibition after trauma was assessed in a subgroup of WT mice pretreated with anti-mouse CD62P (Clone: RB40.34, BD) antibody and in P-selectin-deficient mice. All captured images were analyzed offline for the adhesion and aggregation of platelets and leukocytes and occlusion of cremaster venules. Pre- and post-injury fluorescence intensity of platelets and leukocytes were compared by t-test, and the changes in these signals induced by trauma were compared between different experimental groups of mice using ANOVA with Dunnett's pairwise comparisons and significance at p<0.05. Results: At baseline, WT mice showed very few leukocytes rolling on cremaster venules and no adherent leukocytes or platelets in either arterioles or venules. Immediately following trauma, leukocyte rolling and firm adhesion to the cremaster venules were markedly increased, followed by platelet adhesion. Adherent platelets and leukocyte aggregates continued to accumulate and form multiple growing thrombi, which ultimately occluded the venules by 15 minutes post-trauma. Notably, no fibrin was detected in these venular thrombi. P-selectin blockade in the WT mice reduced the number of rolling leukocytes within a few minutes of treatment; no rolling leukocytes were observed in venules of P-selectin-deficient mice at baseline. After trauma, both P-selectin-inhibited and P-selectin-deficient mice had significantly reduced platelet adhesion to the vessel wall and no leukocyte rolling or adhesion. Only a few platelets adhered to the vessel wall, forming small, unstable thrombi. These platelet aggregates failed to grow into larger thrombi and did not occlude vessels, and microcirculatory flow remained uninterrupted. Conclusion(s): Here, we show that microvascular dysfunction that leads to eventual obstruction by platelet–leukocyte thrombi is an almost immediate consequence of polytrauma. Our studies reveal that P-selectin plays a crucial role in this process, as its blockade drastically reduces leukocyte adhesion and platelet thrombosis. Although we have yet to delineate the precise molecular mechanisms, this is a previously undescribed role for P-selectin and identifies it as a promising target in trauma therapy. P-selectin blockade is likely to have limited or no effect on the hemostatic response to the injury but a good chance of preventing or ameliorating downstream sequelae.