Platelet Biochemistry and Morphology after Cryopreservation

Platelet cryopreservation has been investigated for several decades as an alternative to room temperature storage of platelet concentrates. The use of dimethylsulfoxide as a cryoprotectant has improved platelet storage and cryopreserved concentrates can be kept at −80 °C for two years. Cryopreserved platelets can serve as emergency backup to support stock crises or to disburden difficult logistic areas like rural or military regions. Cryopreservation significantly influences platelet morphology, decreases platelet activation and severely abrogates platelet aggregation. Recent data indicate that cryopreserved platelets have a procoagulant phenotype because thrombin and fibrin formation kicks in earlier compared to room temperature stored platelets. This happens both in static and hydrodynamic conditions. In a clinical setting, low 1-h post transfusion recoveries of cryopreserved platelets represent fast clearance from circulation which may be explained by changes to the platelet GPIbα receptor. Cryopreservation splits the concentrate in two platelet subpopulations depending on GPIbα expression levels. Further research is needed to unravel its physiological importance. Proving clinical efficacy of cryopreserved platelets is difficult because of the heterogeneity of indications and the ambiguity of outcome measures. The procoagulant character of cryopreserved platelets has increased interest for use in trauma stressing the need for double-blinded randomized clinical trials in actively bleeding patients

Microfluidic flow chambers using reconstituted blood to model hemostasis and platelet transfusion in vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion

A comparison of haematopoietic stem cells from umbilical cord blood and peripheral blood for platelet production in a microfluidic device

Background and objectives: Several sources of haematopoietic stem cells have been used for static culture of megakaryocytes to produce platelets in vitro. This study compares and characterizes platelets produced in shear flow using precursor cells from either umbilical (UCB) or adult peripheral blood (PB). Materials and methods: The efficiency of platelet production of the cultured cells was studied after perfusion in custom-built von Willebrand factor-coated microfluidic flow chambers. Platelet receptor expression and morphology were investigated by flow cytometry and microscopy, respectively. Results: Proliferation of stem cells isolated out of UCB was significantly higher (P < 0 center dot 0001) compared to PB. Differentiation of these cells towards megakaryocytes was significantly lower from PB compared to UCB where the fraction of CD42b/CD41 double positive events was 44 +/- 9% versus 76 +/- 11%, respectively (P < 0 center dot 0001). However, in vitro platelet production under hydrodynamic conditions was more efficient with 7 center dot 4 platelet-like particles per input cell from PB compared to 4 center dot 2 from UCB (P = 0 center dot 02). The percentage of events positive for CD42b, CD41 and CD61 was comparable between both stem cell sources. The mean number of receptors per platelet from UCB and PB was similar to that on blood bank platelets with on average 28 000 CD42b, 57 000 CD61 and 5500 CD49b receptors. Microscopy revealed platelets appearing similar to blood bank platelets in morphology, size and actin cytoskeleton, alongside smaller fragments and source megakaryocytes. Conclusion: This characterization study suggests that platelets produced in vitro under flow either from UCB or from PB share receptor expression and morphology with donor platelets stored in the blood bank

Biomolecular consequences of platelet pathogen inactivation methods

Pathogen inactivation (PI) for platelet concentrates (PC) is a fairly recent development in transfusion medicine that is intended to decrease infectious disease transmission from the donor to the receiving patient Effective inactivation of viruses, bacteria and eukaryotic parasites adds a layer of safety, protecting the blood supply against customary and emerging pathogens. Three PI methods have been described for platelets. These are based on photochemical damage of nucleic acids which prevents replication of most infectious pathogens and contaminating donor leukocytes. Because platelets do not replicate, the collateral damage to platelet function is considered low to non-existing. This is disputable however because photochemistry is not specific for nucleic acids and significantly affects platelet biomolecules as well. The impact of these biomolecular changes on platelet function and hemostasis is not well understood, but is increasingly being studied. The results of these studies can help explain current and future clinical observations with PI platelets, including the impact on transfusion yield and bleeding. This review summarizes the biomolecular effects of PI treatment on platelets. We conclude that despite a comparable principle of photochemical inactivation, all three methods affect platelets in different ways. This knowledge can help blood banks and transfusion specialists to guide their choice when considering the implementation or clinical use of PI treated platelets

Psoralen and ultraviolet A light treatment directly affects phosphatidylinositol 3-kinase signal transduction by altering plasma membrane packing

Psoralen and ultraviolet A light (PUNTA) are used to kill pathogens in blood products and as a treatment of aberrant cell proliferation in dermatitis, cutaneous T-cell lymphoma, and graft versus-host disease. DNA damage is well described, but the direct effects of PUVA on cell signal transduction are poorly understood. Because platelets are anucleate and contain archetypal signal transduction machinery, they are ideally suited to address this. Lipidomics on platelet membrane extracts showed that psoralen forms adducts with unsaturated carbon bonds of fatty acyls in all major phospholipid classes after PUVA. Such adducts increased lipid packing as measured by a blue shift of an environment-sensitive fluorescent probe in model liposomes. Furthermore, the interaction of these liposomes with lipid order-sensitive proteins like amphipathic lipid-packing sensor and a-synuclein was inhibited by PUVA. In platelets, PUVA caused poor membrane binding of Akt and Bruton's tyrosine kinase effectors following activation of the collagen glycoprotein VI and thrombin protease-activated receptor (PAR) 1. This resulted in defective Akt phosphorylation despite unaltered phosphatidylinositol 3,4,5-trisphosphate levels. Downstream integrin activation was furthermore affected similarly by PUVA following PAR1 (effective half-maximal concentration (EC), 8.4 +/- 1.1 versus 4.3 +/- 1.1 mu M) and glycoprotein VI (EC50, 1.61 +/- 0.85 versus 0.26 +/- 0.21 mu g/ml) but not PAR4 (EC50, 50 +/- 1 versus 58 +/- 1 mu m) signal transduction. Our findings were confirmed in T-cells ftom graft-versus-host disease patients treated with extracorporeal photopheresis, a form of systemic PUVA. In conclusion, PUVA increases the order of lipid phases by covalent modification of phospholipids, thereby inhibiting membrane recruitment of effector kinases

Ultraviolet C light pathogen inactivation treatment of platelet concentrates preserves integrin activation but affects thrombus formation kinetics on collagen in vitro

BACKGROUND: Ultraviolet (UV) light illumination in the presence of exogenously added photosensitizers has been used to inactivate pathogens in platelet (PLT) concentrates for some time. The THERAFLEX UV-C system, however, illuminates PLT concentrates with UV-C light without additional photoactive compounds. In this study residual PLT function is measured in a comprehensive paired analysis of UV-C-treated, gamma-irradiated, and untreated control PLT concentrates. STUDY DESIGN AND METHODS: A pool-and-split design was used with buffy coat-derived PLT concentrates in 65% SSP+ additive solution. Thrombus formation kinetics in microfluidic flow chambers onto immobilized collagen was investigated with real-time video microscopy. PLT aggregation, membrane markers, and cellular metabolism were determined concurrently. RESULTS: Compared to gamma-treated and untreated controls, UV-C treatment significantly affected thrombus formation rates on Days 5 and 7, not Day 2. PLT degranulation (P-selectin) and PLT apoptosis (annexin V binding) was slightly but significantly increased from Day 2 on. UV-C treatment moreover induced integrin αIIb β3 conformational changes reminiscent of activation. However, subsequent integrin activation by either PAR1-activating hexapeptide (PAR1AP) or convulxin was unaffected. This was confirmed by PLT aggregation studies induced with collagen, PAR1AP, and ristocetin at two different agonist concentrations. Finally, UV-C slightly increased lactic acid production rates, resulting in significantly decreased pH on Days 5 and 7, but never dropped below 7.2. CONCLUSION: UV-C pathogen inactivation treatment slightly but significantly increases PLT activation markers but does not profoundly influence activatability nor aggregation. The treatment does, however, attenuate thrombus formation kinetics in vitro in microfluidic flow chambers, especially after storage

The contribution of von Willebrand factor-GPIbα interactions to persistent aggregate formation in apheresis platelet concentrates

BACKGROUND AND OBJECTIVES: Apheresis platelet concentrates sometimes contain persistent aggregates (PA). Because apheresis involves extracorporeal circulation, we hypothesized that interactions between GPIbα and von Willebrand factor (VWF) underlie their origin. MATERIALS AND METHODS: Platelets in donations with PA were compared to aggregate-free (AF) controls. Flow cytometry was used to determine platelet bound VWF. Degranulation was measured using P-selectin expression in flow cytometry and cytokine release using immunosorbent assays. Platelet adhesion to VWF was assessed in hydrodynamic flow and real-time video microscopy. RESULTS: Platelets in PA concentrates had significantly more (P = 0·009, n ≥ 8) bound VWF compared to AF platelets, but differences in VWF concentration, VWF collagen binding, activated VWF or GPIbα expression were not found. Degranulation was higher (P = 0·030, n = 7) in PA than AF concentrates on day 1 of storage, but adhesion to immobilized VWF under hydrodynamic flow conditions was normal at that moment. On day 6, however, significantly less VWF adhesion (P = 0·009, n ≥ 6) was found for PA platelets compared to AF, indicating accelerated storage lesion in PA products. In a model that mimicks PA formation by chemically induced binding of VWF to platelets, we found that degranulation, phosphatidylserine expression and metabolism did not differ with paired controls at any time during subsequent storage. CONCLUSION: Accelerated storage lesion is found in concentrates with PA, but this cannot be explained solely by increased platelet bound VWF following apheresis. Therefore, additional stressors are probably responsible for the increases observed in platelet degranulation and storage lesion in products with PA

Persistent aggregates in apheresis platelet concentrates are commonly collected from donors with a history of aggregate donation

Platelet apheresis sometimes causes persistent aggregates (PA). This study (n = 211) shows that changing the apheresis settings to reach fixed product volumes instead of yields does not influence PA incidence, even though PA products on average contain more platelets than controls. Furthermore, logistic regression was used to model if PA can be predicted on the basis of certain predonation parameters. PA donation history was the only parameter retained, proving a strong determinant of predictability [AUC = 0.735 (SE = 0.022)]. Consequently, donations from a donor with previous PA history are 7.8 times more likely to contain PA than from a donor without preceding history

A microfluidic flow chamber model for platelet transfusion and hemostasis measures platelet deposition and fibrin formation in real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay