D-Dimer and Fibrin Degradation Products Impair Platelet Signaling: Plasma D-Dimer Is a Predictor and Mediator of Platelet Dysfunction During Trauma.

BACKGROUND: Platelet dysfunction often accompanies trauma-induced coagulopathy. Because soluble fibrin impairs platelet glycoprotein VI (GPVI) signaling and platelets of trauma patients can display impaired calcium mobilization, we explored the role of fibrinolysis on platelet dysfunction during trauma. METHODS: Convulxin-induced GPVI calcium mobilization was investigated in healthy platelet-rich plasma (PRP) pretreated with thrombin and tissue plasminogen activator (tPA). Blood samples from healthy participants (n = 7) and trauma patients (n = 22) were tested for platelet calcium mobilization, plasma D-dimer, platelet D-dimer binding (via flow cytometry), and platelet lumi-aggregometry. RESULTS: For healthy platelets, maximal platelet dysfunction was observed when cross-linked soluble fibrin (no tPA) or cross-linked fibrin degradation products (FDPs) were generated in suspension before convulxin stimulation. Lack of fibrin polymerization (inhibited by Gly-Pro-Arg-Pro [GPRP]) or lack of factor XIIIa cross-linking (T101-inhibited) restored GPVI signaling, whereas non-cross-linked FDPs only partially blocked signaling induced by convulxin. In addition, D-dimer added to healthy PRP impaired platelet aggregation and dense granule release induced by various agonists. Plasma D-dimer level was strongly correlated (R = 0.8236) with platelet dysfunction as measured by platelet calcium mobilization induced with various agonists. By 48 to 120 h after trauma, plasma D-dimer levels declined, and platelet function increased significantly but not to healthy levels. Trauma platelets displayed elevated D-dimer binding that was only partially reduced by αIIbß3-inhibitor GR144053. After 60-minute incubation, washed healthy platelets resuspended in plasma from trauma patients captured approximately 10 000 D-dimer equivalents per platelet. CONCLUSIONS: During trauma, D-dimer and FDPs inhibit platelets, potentially via GPVI and integrin αIIbß3 engagement, contributing to a fibrinolysis-dependent platelet loss-of-function phenotype.

Platelet dysfunction during trauma involves diverse signaling pathways and an inhibitory activity in patient-derived plasma.

BACKGROUND: Trauma-induced coagulopathy occurs in about 25% of injured patients and accounts for about 10% of deaths worldwide. Upon injury, hemostatic function may decline due to vascular dysfunction, clotting factor deficiencies, hyperfibrinolysis, and/or platelet dysfunction. We investigated agonist-induced calcium signaling in platelets obtained over time from trauma patients. METHODS: Platelets from trauma patients and healthy donors were monitored via intracellular calcium mobilization and flow cytometry markers (α2bß3 activation, P-selectin display, and phosphatidylserine exposure) following stimulation with a panel of agonists (adenosine 5'-diphosphate sodium salt, U46619, convulxin, PAR-1/4 activating peptides, iloprost) used in isolation or in pairwise tests. Furthermore, healthy donor platelets were tested in heterologous plasma isolated from healthy subjects and trauma patients. RESULTS: When exposed to agonists over the first 24 hours postinjury, trauma patient platelets mobilized less calcium in comparison to healthy platelets. Partial recovery of platelet activity was observed in about a third of patients after 120 hours, although not fully obtaining healthy baseline function. Flow cytometry markers of trauma platelets were similar to healthy platelets prior to stimulation, but were depressed in trauma platelets stimulated with adenosine 5'-diphosphate sodium salt or convulxin. Also, washed healthy platelets showed a significant reduction in calcium mobilization when reconstituted in plasma from trauma patients, relative to healthy plasma, at all plasma doses tested. CONCLUSION: Platelet dysfunction in trauma patients included poor response to multiple agonists relevant to hemostatic function. Furthermore, the inhibitor effect of patient plasma on healthy platelets suggests that soluble plasma species may downregulate endogenous or transfused platelets during trauma.

Dual antiplatelet and anticoagulant (APAC) heparin proteoglycan mimetic with shear-dependent effects on platelet-collagen binding and thrombin generation.

Heparin proteoglycans (HEP-PGs) carry standard heparin-mediated anticoagulant properties as well as novel antiplatelet functions, a combination that may be significant for targeting multiple pathways in a single therapy. Recent work developing semisynthetic HEP-PG mimetics has shown promising results also in vivo, however flow conditions in vitro that replicate in vivo hemodynamics have not been reported. In this work, we present several assays (platelet calcium mobilization, aggregometry, microfluidic tests at venous and arterial hemodynamics) to characterize specific mechanistic effects of dual antiplatelet and anticoagulant (APAC) constructs as mimetics of HEP-PGs. Three APACs with different conjugation levels of heparin chains (CL10, CL18, HICL) were shown to decrease platelet deposition to collagen surfaces in PPACK-treated whole blood at venous shear rate (200 s-1). FXIIa-inhibited whole blood (CTI: corn trypsin inhibitor, 40 µg/mL) perfused over collagen/tissue factor showed reduced both platelet and fibrin deposition when treated with APACs. IC50 values for platelet and fibrin inhibition were calculated for each molecule at venous shear rate. Increasing the shear rate to arterial flows (1000 s-1) and using APAC as the sole anticoagulant, resulted in a more potent antiplatelet effect of APAC, suggesting an added effect on von Willebrand Factor (vWF) function. Additionally, APAC caused an inhibition of calcium mobilization specific to thrombin and collagen stimulation and a dose-dependent reduction in collagen-mediated platelet aggregation. Understanding the sensitivity of APAC activity to shear rate, platelet signaling and procoagulant pathways is important for applications in which APAC administration may have beneficial therapeutic effects.