The Effect of Hematocrit on Platelet Adhesion: Experiments and Simulations.

The volume fraction of red blood cells (RBCs) in a capillary affects the degree to which platelets are promoted to marginate to near a vessel wall and form blood clots. In this work we investigate the relationship between RBC hematocrit and platelet adhesion activity. We perform experiments flowing blood samples through a microfluidic channel coated with type 1 collagen and observe the rate at which platelets adhere to the wall. We compare these results with three-dimensional boundary integral simulations of a suspension of RBCs and platelets in a periodic channel where platelets can adhere to the wall. In both cases, we find that the rate of platelet adhesion varies greatly with the RBC hematocrit. We observe that the relative decrease in platelet activity as hematocrit falls shows a similar profile for simulation and experiment.

Electron Microscopy as a Tool for Assessment of Anticoagulation Strategies During Extracorporeal Life Support: The Proof Is on the Membrane.

Extracorporeal life support (ECLS) is fast becoming more common place for use in adult patients failing mechanical ventilation. Management of coagulation and thrombosis has long been a major complication in the use of ECLS therapies. Scanning electron microscopy (SEM) of membrane oxygenators (MOs) after use in ECLS circuits can offer novel insight into any thrombotic material deposition on the MO. In this pilot study, we analyzed five explanted MOs immediately after use in a sheep model of different acute respiratory distress syndrome (ARDS). We describe our methods of MO dissection, sample preparation, image capture, and results. Of the five MOs analyzed, those that received continuous heparin infusion showed very little thrombosis formation or other clot material, whereas those that were used with only initial heparin bolus showed readily apparent thrombotic material.

Rapid assessment of shock in a nonhuman primate model of uncontrolled hemorrhage: Association of traditional and nontraditional vital signs to mortality risk.

BACKGROUND: Heart rate (HR), systolic blood pressure (SBP) and mean arterial pressure (MAP) are traditionally used to guide patient triage and resuscitation; however, they correlate poorly to shock severity. Therefore, improved acute diagnostic capabilities are needed. Here, we correlated acute alterations in tissue oxygen saturation (StO2) and end-tidal carbon dioxide (ETCO2) to mortality in a rhesus macaque model of uncontrolled hemorrhage. METHODS: Uncontrolled hemorrhage was induced in anesthetized rhesus macaques by a laparoscopic 60% left-lobe hepatectomy (T = 0 minute). StO2, ETCO2, HR, as well as invasive SBP and MAP were continuously monitored through T = 480 minutes. At T = 120 minutes, bleeding was surgically controlled, and blood loss was quantified. Data analyses compared nonsurvivors (expired before T = 480 minutes, n = 5) with survivors (survived to T = 480 minutes, n = 11) using repeated-measures analysis of variance with Bonferroni correction. All p < 0.05 was considered statistically significant. Results were reported as mean ± SEM. RESULTS: Baseline values were equivalent between groups for each parameter. In nonsurvivors versus survivors at T = 5 minutes, StO2 (55% ± 10% vs. 78% ± 3%, p = 0.02) and ETCO2 (15 ± 2 vs. 25 ± 2 mm Hg, p = 0.0005) were lower, while MAP (18 ± 1 vs. 23 ± 2 mm Hg, p = 0.2), SBP (26 ± 2 vs. 34 ± 3 mm Hg, p = 0.4), and HR (104 ± 13 vs. 105 ± 6 beats/min, p = 0.3) were similar. Association of values over T = 5-30 minutes to mortality demonstrated StO2 and ETCO2 equivalency with a significant group effect (p ≤ 0.009 for each parameter; R(2) = 0.92 and R(2) = 0.90, respectively). MAP and SBP associated with mortality later into the shock period (p < 0.04 for each parameter; R(2) = 0.91 and R(2) = 0.89, respectively), while HR yielded the lowest association (p = 0.8, R(2) = 0.83). CONCLUSION: Acute alterations in StO2 and ETCO2 strongly associated with mortality and preceded those of traditional vital signs. The continuous, noninvasive aspects of Food and Drug Administration-approved StO2 and ETCO2 monitoring devices provide logistical benefits over other methodologies and thus warrant further investigation.

Both acute delivery of and storage with magnesium sulfate promote cold-stored platelet aggregation and coagulation function.

BACKGROUND: The platelet storage lesion causes loss of function and viability over time. A new paradigm for platelet storage is desired to enable safer, more effective transfusions while reducing waste. We hypothesized that repletion of Mg, which is chelated by citrate anticoagulant, could reduce platelet storage lesion severity when given in conjunction with storage at a refrigerated temperature. METHODS: Apheresis platelet units were collected from healthy donors and stored at 22°C or 4°C. On Days 0, 2, 4, and 8, samples were collected for analyses of receptor-mediated aggregation, coagulation, adhesion to collagen under flow, and viability. In the first series, samples were given an acute dose of MgSO4 before testing; in the second series, storage bags were supplemented with 0-, 3-, or 6-mM MgSO4. RESULTS: Acutely delivered MgSO4 induced a more rapid coagulation time in apheresis platelets, further enhanced by storage at 4°C. Platelet adhesion to a collagen surface while exposed to arterial shear rates (920 s) was enhanced by MgSO4 supplementation-acute MgSO4 had a large effect on adhesion of fresh platelets, which diminished more rapidly in 22°C samples, while storage with MgSO4 showed significant benefits even out to Day 4 at both temperatures. Although 4°C storage improves the longevity of platelet aggregation responses to agonists, MgSO4 supplementation did not change those responses. CONCLUSION: Acute MgSO4 reduces clot time likely through the transient increase of free Ca. Limited differences between platelet function in acute delivery of and storage with MgSO4 diminish the possibility that Mg-induced metabolic inhibition of platelets synergizes with 4°C storage. Regardless, magnesium supplementation to platelets is an exciting possibility in transfusion because the adhesion response of 22°C-stored platelets on Day 4 is significantly enhanced when stored with 6-mM MgSO4.

Comparative response of platelet fV and plasma fV to activated protein C and relevance to a model of acute traumatic coagulopathy.

BACKGROUND: Acute traumatic coagulopathy (ATC) has been linked to an increase in activated protein C (aPC) from 40 pM in healthy individuals to 175 pM. aPC exerts its activity primarily through cleavage of active coagulation factor Va (fVa). Platelets reportedly possess fVa which is more resistant to aPC cleavage than plasma fVa; this work examines the hypothesis that normal platelets are sufficient to maintain coagulation in the presence of elevated aPC. METHODS: Coagulation responses of normal plasma, fV deficient plasma (fVdp), and isolated normal platelets in fVdp were conducted: prothrombin (PT) tests, turbidimetry, and thromboelastography (TEG), including the dose response of aPC on the samples. RESULTS: PT and turbidimetric assays demonstrate that normal plasma is resistant to aPC at doses much higher than those found in ATC. Additionally, an average physiological number of washed normal platelets (200,000 platelets/mm3) was sufficient to eliminate the anti-coagulant effects of aPC up to 10 nM, nearly two orders of magnitude above the ATC concentration and even the steady-state pharmacological concentration of human recombinant aPC, as measured by TEG. aPC also demonstrated no significant effect on clot lysis in normal plasma samples with or without platelets. CONCLUSIONS: Although platelet fVa shows slightly superior resistance to aPC's effects compared to plasma fVa in static models, neither fVa is sufficiently cleaved in simulations of ATC or pharmacologically-delivered aPC to diminish coagulation parameters. aPC is likely a correlative indicator of ATC or may play a cooperative role with other activity altering products generated in ATC.

Cellular regulation of blood coagulation: a model for venous stasis.

We have adapted the corn-trypsin inhibitor whole-blood model to include EA.hy926 as an endothelium surrogate to evaluate the vascular modulation of blood coagulation initiated by relipidated recombinant tissue factor (rTf) and a cellular Tf surrogate, lipopolysaccharide (LPS)-stimulated THP1 cells (LPS-THP-1). Compared with bare tubes, EA.hy926 with rTf decreased the rate of thrombin formation, ITS accumulation, and the production of fibrinopeptide A. These phenomena occurred with increased rates of factor Va (fVa) inactivation by cleavages at R(506) and R(306). Thus, EA.hy926 provides thrombin-dependent protein C activation and APC fVa inactivation. Comparisons of rTf with LPS-THP-1 showed that the latter gave reduced rates for TAT formation but equivalent fibrinopeptide A, and fV activation/inactivation. In the presence of EA.hy926, the reverse was obtained; with the surrogate endothelium and LPS-THP-1 the rates of TAT generation, fibrinopeptide release, and fV activation were almost doubled, whereas cleavage at R(306) was equivalent. These observations suggest cooperativity between the 2 cell surrogates. These data suggest that the use of these 2 cell lines provides a reproducible quasi-endothelial quasi-inflammatory cytokine-stimulated monocyte system that provides a method to evaluate the variations in blood phenotype against the background of stable inflammatory cell activator and a stable vascular endothelial surrogate.

Postresuscitation tissue neutrophil infiltration is time-dependent and organ-specific.

BACKGROUND: Hemorrhagic shock with conventional resuscitation (CR) primes circulating neutrophils and activates vascular endothelium for increased systemic inflammation, superoxide release, and end-organ damage. Adjunctive direct peritoneal resuscitation (DPR) with intraperitoneal instillation of a clinical peritoneal dialysis solution decreases systemic inflammation and edema formation by enhancing tissue perfusion. The aim of this study is to determine the effect of adjunctive DPR on neutrophil and fluid sequestration. METHODS: Anesthetized rats were hemorrhaged to 40% mean arterial pressure for 60 min. Animals were randomized for CR with the return of the shed blood plus two volumes of saline, or CR plus adjunctive DPR with 30 mL of intraperitoneal injection of a clinical peritoneal dialysis solution. Tissue myeloperoxidase (MPO) level, a marker of neutrophil sequestration, and total water content were assessed in the gut, lung, and liver in sham animals and at time-points 1, 2, 4, and 24 h postresuscitation. RESULTS: Resuscitation from hemorrhagic shock increases MPO level in all tissues in a near-linear fashion during the first 4 h following resuscitation. This occurs irrespective of the resuscitation regimen used. Tissue MPO level returned to baseline at 24 h following resuscitation except in the liver where CR and not adjunctive DPR caused a significant rebound increase. Adjunctive DPR prevented the CR-mediated obligatory fluid sequestration in the gut and lung and maintained a relative normal tissue water in these organs compared with CR alone (n = 7, F = 10.1, P < 0.01). CONCLUSION: Hemorrhagic shock and resuscitation produces time-dependent organ-specific trends of neutrophil sequestration as measured with tissue levels of myeloperoxidase, a marker of neutrophil infiltration. Modulation of the splanchnic blood flow by direct peritoneal resuscitation did not alter the time-dependent neutrophil infiltration in end-organs, suggesting a subordinate role of blood rheology in the hemorrhage-induced neutrophil sequestration. Vulnerable window for neutrophil-mediated tissue damage exists during the first 4 h following resuscitation from hemorrhagic shock in rats. Direct peritoneal resuscitation prevents the early obligatory fluid sequestration and promotes early fluid mobilization.

Clinical peritoneal dialysis solutions modulate white blood cell-intestinal vascular endothelium interaction.

BACKGROUND: Hemorrhagic shock (HS) with conventional resuscitation (CR) (HSCR) primes neutrophils and modulates leukocyte (WBC)-endothelium interaction as part of an exaggerated systemic inflammatory response. We hypothesize that topical application of clinical peritoneal dialysis solutions (PD) modulates such interaction. METHODS: Intestinal intravital microscopy was used to measure WBC rolling in terminal ileum post capillary venules (V2 and V3) in sham-operated animals, and in animals that underwent fixed pressure hemorrhage (50% mean arterial pressure for 60 minutes), followed by conventional resuscitation with the return of the shed blood and 2 vol of saline. Number of rolling WBCs per thirty seconds in selected V2 and V3, bathed in either Kreb's solution or a 2.5% clinical peritoneal dialysis solution (PD) was quantified. Diameters were measured for the in-flow arterioles (A1), and out-flow venules (V1), for calculation of local blood flow with optical Doppler velocimetry. RESULTS: The PD solution significantly (P < .05, n = 11) attenuated WBC-endothelium interaction in sham-operated animals while no significant difference was elicited in HSCR (P > .05, n = 9 Kreb's, n = 7 PD). In addition, the PD solution produced an instantaneous dilation at all levels of the intestinal arterioles in both sham and HSCR. While intestinal venular blood outflow was increased by the PD solution, venular diameters changed very little. CONCLUSION: Superfusion of the gut with glucose-based peritoneal dialysis solutions decreases the concentration of rolling leukocytes along the venular vascular endothelium by a vasodilation-mediated increase in arteriolar inflow and venous outflow mechanism. Hemorrhagic shock and conventional resuscitation enhance the concentration of rolling leukocytes presumably by mechanisms related to upregulation of the adhesion molecules and the low-flow state. Hemorrhage and resuscitation-enhanced leukocytes rolling was not reversed by adjunctive DPR despite the associated marked increase in arterial inflow and venous outflow. The status of the endothelium and the level of leukocyte priming in low-flow states are stronger predictors of leukocyte-endothelium interaction than rheology factors.