Platelets stored at 4°C contribute to superior clot properties compared to current standard-of-care through fibrin-crosslinking.

Currently, platelets for transfusion are stored at room temperature (RT) for 5-7 days with gentle agitation, but this is less than optimal because of loss of function and risk of bacterial contamination. We have previously demonstrated that cold (4°C) storage is an attractive alternative because it preserves platelet metabolic reserves, in vitro responses to agonists of activation, aggregation and physiological inhibitors, as well as adhesion to thrombogenic surfaces better than RT storage. Recently, the US Food and Drug Administration clarified that apheresis platelets stored at 4°C for up to 72 h may be used for treating active haemorrhage. In this work, we tested the hypothesis that cold-stored platelets contribute to generating clots with superior mechanical properties compared to RT-stored platelets. Rheological studies demonstrate that the clots formed from platelets stored at 4°C for 5 days are significantly stiffer (higher elastic modulus) and stronger (higher critical stress) than those formed from RT-stored platelets. Morphological analysis shows that clot fibres from cold-stored platelets were denser, thinner, straighter and with more branch points or crosslinks than those from RT-stored platelets. Our results also show that the enhanced clot strength and packed structure is due to cold-induced plasma factor XIII binding to platelet surfaces, and the consequent increase in crosslinking.

Procoagulant activity of human mesenchymal stem cells.

BACKGROUND: Allogeneic mesenchymal stem cells (MSCs) show great potential for the treatment of military and civilian trauma based on their reduced immunogenicity and ability to modulate inflammation and immune function in the recipient. Although generally considered to be safe, MSCs express tissue factor (TF), a potent activator of coagulation. In the current study, we evaluated multiple MSC populations for tissue factor expression and procoagulant activity to characterize safety considerations for systemic use of MSCs in trauma patients who may have altered coagulation homeostasis. METHODS: Multiple MSC populations derived from either human adipose tissue or bone marrow were expanded in the recommended stem cell media. Stem cell identity was confirmed using a well-characterized panel of positive and negative markers. Tissue factor expression on the cell surface was evaluated by flow cytometry with anti-CD142 antibody. Effects on blood coagulation were determined by thromboelastography and calibrated automated thrombogram assays using platelet-poor plasma or whole blood. RESULTS: Mesenchymal stem cells express tissue factor on their surfaces and are procoagulant in the presence of blood or plasma. The adipose-derived MSCs (Ad-MSC) evaluated were more procoagulant and expressed more tissue factor than bone marrow MSCs (BM-MSCs), which showed a greater variability in TF expression. Bone marrow MSCs were identified that exhibited low procoagulant activity, whereas all Ad-MSCs examined exhibited high procoagulant activity. The percentage of cells in a given population expressing surface tissue factor correlates roughly with functional procoagulant activity. Mesenchymal stem cell tissue factor expression and procoagulant activity change over time in culture. CONCLUSIONS: All MSC populations are not equivalent; care should be taken to select cells for clinical use that minimize potential safety problems and maximize chance of patient benefit. Adipose-derived MSCs seem more consistently procoagulant than BM-MSCs, presenting a potential safety concern for systemic administration in coagulopathic patients. Donor variation exists between different cell populations, and culture handling conditions may also determine coagulation activity. Cells must be routinely monitored during preparation to ensure that they retain the desired characteristics before patient administration.

Acute traumatic coagulopathy: The elephant in a room of blind scientists.

Acute traumatic coagulopathy (ATC) is the failure of coagulation homeostasis that can rapidly arise following traumatic injury, hemorrhage, and shock; it is associated with higher injury severity, coagulation abnormalities, and increased blood transfusions. Acute traumatic coagulopathy has historically been defined by a prolonged prothrombin time, although newer, more informative measurements of hemostatic function have been used to improve diagnosis and support clinical decision making. The underlying biochemical mechanisms of and best practice therapeutics for ATC remain under active investigation because of its significant correlation to poor outcomes. The wide range of hypothesized mechanisms for ATC results from the large number of symptoms, phenotypes, and altered states in these patients as observed by multiple research groups. Much like the ancient fable of blind men describing an elephant from their limited perspectives, the limited nature of clinical and laboratory tools used to diagnose coagulopathy or evaluate hemostatic function has made finding causation difficult. The prolonged prothrombin time, degree of fibrinolysis, depletion of coagulation factors and inhibitors, and general failure of the blood have all been identified as being primary indicators for ATC. Therapeutic interventions including recombinant coagulation factors, antifibrinolytics, and blood products have been used with varying degrees of success as they are used to address specific symptoms. To truly understand the causes of ATC, research efforts must recognize the complexity of the hemostatic system and get to the heart of the matter by answering the question: "Is ATC a pathological condition that develops from the observed deficiencies in coagulation, fibrinolysis, and autoregulation, or is ATC an adaptive response generated as the body attempts to restore perfusion and avoid massive organ failure?" Because patient management must proceed without definitive answers regarding the entire causative chain, the current therapeutic focus should be on using what knowledge has been gained to the patient's advantage: control hemorrhage, maintain appropriate homeostatic balances of coagulation proteins, and restore oxygen perfusion.

ENDOTHELIUM-DERIVED INHIBITORS EFFICIENTLY ATTENUATE THE AGGREGATION AND ADHESION RESPONSES OF REFRIGERATED PLATELETS.

Refrigeration of platelets (4°C) provides the possibility of improving transfusion practice over the current standard-of-care, room temperature (RT) storage. However, the increased level of platelet activation observed at 4°C in vitro is cause for concern of uncontrolled thrombosis in vivo. In this study, we assessed the safety of 4°C-stored platelets by evaluating their response to physiologic inhibitors prostacyclin (PGI2) and nitric oxide (NO). Apheresis platelets were collected from healthy donors (n = 4) and tested on Day 1 (fresh) or Day 5 (RT- and 4°C-stored) after treatment with PGI2 and NO or not for: thrombin generation; factor V (FV) activity; intracellular free calcium, cAMP and cGMP; ATP release; TRAP-induced activation; aggregation to ADP, collagen, and TRAP, and adhesion to collagen under arterial flow. Data were analyzed using two-way ANOVA and post-hoc Tukey test for multiple comparisons, with significance set at P < 0.05. Treatment with inhibitors increased intracellular cAMP and cGMP levels in fresh and stored platelets. Thrombin generation was significantly accelerated in stored platelets consistent with increased factor V levels, PS exposure, CD62P expression, intracellular free calcium, and ATP release. While treatment with inhibitors did not attenuate thrombin generation in stored platelets, activation, aggregation, and adhesion responses were inhibited by both PGI2 and NO in 4°C-stored platelets. In contrast, though RT-stored platelets were activated, they did not adhere or aggregate in response to agonists. Thus, refrigerated platelets maintain their intracellular machinery, are responsive to agonists and platelet function inhibitors, and perform hemostatically better than RT-stored platelets.

Hemostatic function of apheresis platelets stored at 4°C and 22°C.

INTRODUCTION: Platelet refrigeration decreases the risk of bacterial contamination and may preserve function better than standard-of-care room temperature (RT) storage. Benefits could include lower transfusion-related complications, decreased costs, improved hemostasis in acutely bleeding patients, and extended shelf life. In this study, we compared the effects of 22°C and 4°C storage on the functional and activation status of apheresis platelets. METHODS: Apheresis platelets (n = 5 per group) were stored for 5 days at 22°C with agitation (RT) versus at 4°C with agitation (4°C + AG) and without (4°C). Measurements included platelet counts, mean platelet volume, blood gas analytes, aggregation response, thromboelastography, thromboxane B2 and soluble CD40 ligand release, activation markers, and microparticle formation. RESULTS: Sample pH levels were within acceptable limits for storage products (pH 6.2-7.4). Platelet glucose metabolism (P < 0.05), aggregation response (adenosine diphosphate: RT 0; 4°C + AG 5.0 ± 0.8; 4°C 5.6 ± 0.9; P < 0.05), and clot strength (maximum amplitude: RT 58 ± 2; 4°C + AG 63 ± 2; 4°C 67 ± 2; P < 0.05) were better preserved at 4°C compared with RT storage. Refrigerated samples were more activated compared with RT (P < 0.05), although thromboxane B2 (P < 0.05) and soluble CD40 ligand release (P < 0.05) were higher at RT. Agitation did not improve the quality of 4°C-stored samples. CONCLUSIONS: Apheresis platelets stored at 4°C maintain more viable metabolic characteristics, are hemostatically more effective, and release fewer proinflammatory mediators than apheresis platelets stored at RT over 5 days. Given the superior bacteriologic safety of refrigerated products, these data suggest that cold-stored platelets may improve outcomes for acutely bleeding patients.

Primary hemostatic capacity of whole blood: a comprehensive analysis of pathogen reduction and refrigeration effects over time.

BACKGROUND: Whole blood (WB) has been used in combat since World War I as it is readily available and replaces every element of shed blood. Component therapy has become standard; however, recent military successes with WB resuscitation have revived the debate regarding wider WB use. Characterization of optimal WB storage is needed. We hypothesized that refrigeration preserves WB function and that a pathogen reduction technology (PRT) based on riboflavin and ultraviolet light has no deleterious effect over 21 days of storage. STUDY DESIGN AND METHODS: WB units were stored for 21 days either at 4°C or 22°C. Half of each temperature group underwent PRT, yielding four final treatment groups (n = 8 each): CON 4 (WB at 4°C); CON 22 (WB at 22°C); PRT 4 (PRT WB at 4°C); and PRT 22 (PRT WB at 22°C). Testing was at baseline, Days 1-7, 10, 14, and 21. Assays included coagulation factors; platelet activation, aggregation, and adhesion; and thromboelastography (TEG). RESULTS: Prothrombin time (PT) and partial thromboplastin time increased over time; refrigeration attenuated the effects on PT (p ≤ 0.009). Aggregation decreased over time (p ≤ 0.001); losses were attenuated by refrigeration (p ≤ 0.001). Refrigeration preserved TEG parameters (p ≤ 0.001) and PRT 4 samples remained within normal limits throughout the study. Refrigeration in combination with PRT inhibited fibrinolysis (p ≤ 0.001) and microparticle formation (p ≤ 0.031). Cold storage increased shear-induced platelet aggregation and ristocetin-induced platelet agglutination (p ≥ 0.032), as well as GPIb-expressing platelets (p ≤ 0.009). CONCLUSION: The in vitro hemostatic function of WB is largely unaffected by PRT treatment and better preserved by cold storage over 21 days. Refrigerated PRT WB may be suitable for trauma resuscitation. Clinical studies are warranted.

Enhanced shear-induced platelet aggregation due to low-temperature storage.

BACKGROUND: Refrigeration of platelets (PLTs) offers an attractive alternative to the currently practiced storage at room temperature since it may mitigate problems associated with bacterial contamination and extend storage lifetime. Refrigeration causes a number of biophysical and biochemical changes in PLTs and decreases PLT circulation time in vivo. However, the effect of refrigeration on PLT hemostatic functions under physiologic and pathophysiologic shear conditions has not been adequately characterized. STUDY DESIGN AND METHODS: Washed PLTs prepared from either fresh PLT-rich plasma (PRP) or PRP stored at 4°C for 2 days was mixed with exogenous von Willebrand factor (VWF) and fibrinogen and sheared in a cone-and-plate viscometer. PLT aggregation, activation, and VWF binding after shear and glycoprotein (GP) Ibα receptor expression and ristocetin-induced PLT agglutination were measured. RESULTS: PLTs stored at 4°C for 2 days aggregated significantly more than fresh PLTs particularly at high shear rates (10,000/sec), and this increase was independent of PLT concentration or suspension viscosity. Further, refrigerated PLTs showed a greater increase in GP Ibα-dependent PLT activation under shear and also bound more VWF than fresh PLTs. However, the GP Ibα expression levels as measured by three different antibodies were significantly lower in refrigerated PLTs than in fresh PLTs, and refrigeration resulted in a modest decrease in ristocetin-induced PLT agglutination. CONCLUSION: The combined results demonstrate that refrigeration increases PLT aggregation under high shear, but not static, conditions and also increases shear-induced VWF binding and PLT activation. Clinically, enhanced shear-induced PLT aggregation due to low temperature storage may be a beneficial strategy to prevent severe bleeding in trauma.