A novel, quantitative clot retraction assay to evaluate platelet function.

Blood platelets are crucial to prevent excessive bleeding following injury to blood vessels. Platelets are crucial for the formation of clots and for clot strength. Platelet activation involves aggregation, attachment to fibrin and clot retraction. Most assays that address platelet function measure platelet aggregation, not clot retraction. Here, we describe a 96-well-based clot retraction assay that requires a relatively short runtime and small sample volume. The assay involves continuous optical density monitoring of platelet-rich plasma that is activated with thrombin. The data can be analyzed using time-series analytical tools to generate quantitative information about different phases of clot formation and clot retraction. The assay demonstrated good repeatability and reproducibility and was robust to different calcium concentrations. Impairment of platelet bioenergetics, actin polymerization, fibrin interaction, and signaling significantly affected clot retraction and was detected and showed good agreement with light transmission aggregometry, suggesting that clot retraction is predictive of platelet function. Using this microplate clot retraction assay, we showed a significant difference in platelets stored in autologous plasma compared with platelet additive solution after 7 days of room temperature storage.

Platelets are cell fragments in the blood that are necessary for clot formation. They are crucial to preventing excessive bleeding following trauma. To form clots, platelets clump (aggregate) and attach to fibrin protein and cells inside the blood vessels to form strong web-like structures. Platelets also contract to pull the edges of the wound close. Most measurements of platelet function involve aggregation. This paper focuses on platelet contraction. Here, we describe a new assay to measure platelets contraction that is repeatable and reproducible. The assay uses standard and common laboratory equipment and can be performed by most laboratory personnel and has the potential to detect clinical pathologies of clot formation. The assay could be developed for bedside patient care where platelet function could be assessed rapidly and assist in the diagnosis of coagulation and platelet disorders.

Cold-stored platelets have better preserved contractile function in comparison with room temperature-stored platelets over 21 days.

Although it is well established that transfusion of platelets in cases of severe bleeding reduces mortality, the availability of platelets is hampered by harsh restrictions on shelf life due to elevated risks of microbial contamination and functional losses with room temperature-stored platelets (RTP) kept at 22°C. In contrast, many recent studies have shown that 4°C cold-stored platelets (CSP) are able to overcome these shortcomings leading to the recent Food and Drug Administration licensure for 14-day stored CSP when conventional platelets are unavailable. This work expands the evidence supporting superiority of CSP function by assaying the less explored platelet-mediated clot retraction of RTP and CSP in either autologous plasma (AP) or platelet additive solution (PAS) for up to 21 days. The results demonstrate that CSP have better preservation of contractile function, exhibiting retraction for up to 21 days in both AP and PAS and forming highly ordered fibrin scaffolds similar to those of fresh platelets. In contrast, RTP stored in AP showed impaired contractile function by Day 5 with no retraction after 10 days, whereas PAS-stored RTP retained contractile function for up to 21 days. Collectively, these findings support extended storage of CSP and suggest that storage in PAS can mitigate functional losses in RTP.

Cold storage of platelets in platelet additive solution maintains mitochondrial integrity by limiting initiation of apoptosis-mediated pathways.

BACKGROUND: Cold storage of platelets in plasma maintains hemostatic function and is an attractive alternative to room temperature platelets (RTPs). We have recently shown that functional differences between cold-stored platelets (CSPs) and RTPs after 5-day storage are associated with mitochondrial respiration and that CSPs in platelet (PLT) additive solution (PAS) can maintain hemostatic function for at least 15 days. STUDY DESIGN AND METHODS: This study tested the hypothesis that cold storage in PAS preserves mitochondrial integrity by reducing PLT apoptosis. CSPs and RTPs in plasma or PAS were stored and assayed for up to 15 days for mitochondrial function and integrity, mitochondrial-associated mRNA transcript expression, apoptotic proteins, and apoptotic flow cytometry metrics. RESULTS: CSP preserved mitochondria-associated mRNA comparable to baseline levels, improved mitochondrial respiration, and minimized depolarization to Day 15. Additionally, CSPs had minimal induction of caspases, preservation of plasma membrane integrity, and low expression of pro-apoptotic Bax. Storage in PAS appeared to be protective for RTPs in some parameters and enhanced the effects of CSPs. CONCLUSION: Mitochondrial function and molecular analyses defined CSP priming as distinctly different from the well-documented RTP storage lesion. While current blood bank storage at room temperature is limited to 5 to 7 days, refrigeration and storage in PAS for up to 15 days may represent an opportunity to enhance inventories and access to PLT hemostatic support for bleeding patients.

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.

Recent advances in use of fresh frozen plasma, cryoprecipitate, immunoglobulins, and clotting factors for transfusion support in patients with hematologic disease.

Hematologic diseases include a broad range of acquired and congenital disorders, many of which affect plasma proteins that control hemostasis and immune responses. Therapeutic interventions for these disorders include transfusion of plasma, cryoprecipitate, immunoglobulins, or convalescent plasma-containing therapeutic antibodies from patients recovering from infectious diseases, as well as concentrated pro- or anticoagulant factors. This review will focus on recent advances in the uses of plasma and its derivatives for patients with acquired and congenital hematologic disorders.

Image-based analysis and simulation of the effect of platelet storage temperature on clot mechanics under uniaxial strain.

Optimal strength and stability of blood clots are keys to hemostasis and in prevention of hemorrhagic or thrombotic complications. Clots are biocomposite materials composed of fibrin network enmeshing platelets and other blood cells. We have previously shown that the storage temperature of platelets significantly impacts clot structure and stiffness. The objective of this work is to delineate the relationship between morphological characteristics and mechanical response of clot networks. We examined scanning electron microscope images of clots prepared from fresh apheresis platelets, and from apheresis platelets stored for 5 days at room temperature or at 4 °C, suspended in pooled plasma. Principal component analysis of nine different morphometric parameters revealed that a single principal component (PC1) can distinguish the effect of platelet storage on clot ultrastructure. Finite element analysis of clot response to uniaxial strain was used to map the spatially heterogeneous distribution of strain energy density for each clot. At modest deformations (25% strain), a single principal component (PC2) was able to predict these heterogeneities as quantified by variability in strain energy density distribution and in linear elastic stiffness, respectively. We have identified structural parameters that are primary regulators of stress distribution, and the observations provide insights into the importance of spatial heterogeneity on hemostasis and thrombosis.

Effect of cold storage on shear-induced platelet aggregation and clot strength.

BACKGROUND: Platelets (PLTs) participate in hemostasis and save lives following trauma. PLTs for transfusion are maintained at room temperature (RT, 22°C), limiting viability to 5 days because of metabolic compromise and high risk of bacterial contamination. RT storage may result in weaker clots, delaying hemorrhage control, whereas cold storage (4°C) could permit longer PLT shelf life and result in a more hemostatic product. In this study, we characterized the effect of storage temperature on shear-induced PLT aggregation, clot formation, and strength. METHODS: PLTs obtained from phlebotomized blood or by apheresis were stored at RT or 4°C for 5 days, and PLT aggregation and clot strength were assessed at 37°C. We studied PLT aggregation at steady and complex patterns of shear rates (500-2,500 per second) by flow cytometry, and the kinetics of clot formation and strength were measured using turbidity and dynamic mechanical analysis, respectively. RESULTS: PLT aggregation was higher in 4°C-stored samples on Day 5 compared with fresh or RT-stored samples at all shear rates tested (fresh vs. 4°C and RT vs. 4°C, p < 0.05). PLTs stored at 4°C for 5 days formed significantly stronger clots compared with fresh or RT-stored samples as quantified by turbidity and elastic moduli measurements (fresh vs. 4°C and RT vs. 4°C, p < 0.05). CONCLUSION: Our results show that cold-stored PLTs are more responsive to aggregation stimuli and form stronger clots, presumably because of thicker fibrin strands. These data suggest that the superior functionality of cold-stored PLTs may support faster hemostasis for acutely bleeding in trauma patients compared with RT-stored PLTs.