Generation of annexin V-positive platelets and shedding of microparticles with stimulus-dependent procoagulant activity during storage of platelets at 4 degrees C.

BACKGROUND: The ability of propyl gallate to activate platelet factor 3 has been determined through the activated partial thromboplastin time, but its effect on phosphatidylserine has not been established. STUDY DESIGN AND METHODS: A novel platelet activator, propyl gallate, was introduced to a study of platelets stored at 4 degrees C. The effects of storage on platelet coagulation activity, on phosphatidylserine, and on the shedding of activated and activable membrane particles (microparticles) were examined by activated plasma clotting time, and the effect on annexin V binding was examined by gated flow cytometry. The ratios of annexin V binding and microparticle shedding in stored platelet samples were compared with those in fresh platelets stimulated with propyl gallate. RESULTS: Microparticle shedding by stored platelets compensated for the diminished procoagulant potential of intact platelets (shown as the total propyl gallate-dependent platelet factor 3 activity), which did not change during prolonged (20-day) storage, but levels of phosphatidylserine confined to microparticles increased dramatically as platelet counts fell. Both annexin V binding and microparticle shedding increased spontaneously with storage and artificially with propyl gallate stimulation. However, at the same level of annexin V binding, stored platelets shed more microparticles than did fresh platelets stimulated with propyl gallate. CONCLUSION: Propyl gallate induces platelet procoagulant activity and annexin V binding. Stored platelets differ from fresh platelets in a lower reactivity to propyl gallate activation and a higher rate of microparticle shedding.

Development of optimal techniques for cryopreservation of human platelets. I. Platelet activation during cold storage (at 22 and 8 degrees C) and cryopreservation.

Using the current blood bank storage conditions at 22 degrees C, the viability and function of human platelets can be maintained for only 5 days. This does not allow for the necessary and extensive banking of platelets needed to treat patients afflicted with thrombocytopenia, a side effect of many invasive surgeries such as cardiopulmonary bypass or bone marrow transplantation. The development of optimal techniques for long-term cryopreservation and banking of human platelets would provide the ability to greatly extend the viable life of the platelet and would fulfill an increasing and urgent need in many clinical applications. To determine the optimal techniques for platelet preservation, the expression of an activation marker, phosphatidylserine, on the platelet membrane during storage at 22 and 8 degrees C as well as during the different freezing preservation processes was examined using flow cytometry and annexin V binding assay. Human platelets were identified by both CD41 and light scatter in flow cytometry. In cryopreservation experiments, effects of the following factors on platelet activation were evaluated: (a) cryoprotective agents (CPAs) type: dimethyl sulfoxide (Me2SO), ethylene glycol (EG), and propylene glycol (PG), (b) CPA concentration ranging from 0 to 3 M, and (c) ending temperatures of a slow cooling process at -1 degrees C/min. Our results demonstrated that (a) approximately 50% of platelets were activated on days 7 and 16 at 22 and 8 degrees C, respectively; (b) platelets were not significantly activated after 30-min exposure to 1 M Me2SO, EG, and PG at 22 degrees C, respectively, and (c) there was a significant difference in cryoprotective efficacy among these three CPAs in preventing platelets from cryoinjury. After being cooled to -10 degrees C, 74% of the cryopreserved platelets survived (nonactivated) in 1 M Me2SO solution, while in 1 M EG and 1 M PG solutions, 62 and 42% of the platelets survived, respectively. Using the information that Me2SO consistently yields higher percentages of nonactivated platelets and does not seem to be cytotoxic to platelets for 30-min exposure time, this was found to be the optimal cryoprotective agent for platelets. In addition, significant Me2SO toxicity to platelets was not noted until Me2SO concentrations exceeded 2 M. Finally, a concentration of 1 M Me2SO proved to be the most effective at all cryopreservation ending temperatures tested (-10, -30, -60, and -196 degrees C). In conclusion, under the present experimental conditions, a storage temperature of 8 degrees C appeared to be much better than 22 degrees C. Although the potential chemical toxicity of 1 M Me2SO, EG, or PG is negligible, 1 M Me2SO was found to be optimum for cryopreservation of human platelets. PG has the least cryoprotective function for low-temperature platelet survival.

Platelet membrane early activation markers during prolonged storage.

The relationship between platelet aging and early markers of membrane activation have not been defined clearly. Activation markers expressed during prolonged storage are similar if not identical to those that appear after exposure to thrombin. Using flow cytometry, we investigated platelet membrane expression of CD62P, CD63, and annexin V binding (i.e., loss of membrane asymmetry) in platelets stored for up to 11 days under standard blood banking conditions. We compared five apheresis platelets to two random donor platelet concentrates, and to one pooled platelet preparation from six single platelet concentrates before and after exposure to thrombin. CD62P, CD63 expression, and annexin V binding increased during storage albeit with different kinetics. The differential increments observed between resting and thrombin (1 unit/ml) activated platelets showed an inverse correlation to storage time for CD62P, CD63, and annexin V binding, which was identical to published survival curves. A difference between apheresis platelets and platelet concentrates was observed only on day 1. Our data indicate that the in vitro platelet reserve activity to thrombin activation mirrors that of radiolabeled platelet survival in vivo and that platelet cross-sectional residual life span can explain their diminished capacity to respond to thrombin as a function of viability.

Peripheral blood stem cell collection in a patient with chronic myelogenous leukemia and a high circulating nucleated red cell fraction.

A high level of circulating nucleated red blood cells (NRBC) in patients with chronic myeloproliferative syndromes could potentially complicate peripheral blood stem cell (PSC) collection. The mononuclear NRBC might comprise a significant fraction of the total mononuclear cells in the final product. We report a successful PSC collection in a patient with more NRBC than WBC in the peripheral blood. A 27-year-old man with chronic myelogenous leukemia underwent eight PSC collection procedures, seven using the Cobe Spectra (Spectra) and one using the Fenwal CS3000 Plus (CS). PSC product manipulations to remove NRBC were unnecessary. As assessed by post-collection NRBC: WBC ratio as a percent of the initial ratio, Spectra selectively harvested mononuclear leukocytes over NRBC. The collected products had a mean NRBC: WBC ratio that was 3.4% of the peripheral blood ratio. Adequate numbers of mononuclear leukocytes were collected with less than 6% NRBC contamination. The single CS procedure resulted in a comparable NRBC reduction efficiency as the Spectra. We conclude that PSC harvest using automated blood cell separators from patients with a high level of circulating NRBC may result in a product with an acceptable number of NRBC.

Effect of freezing on the in vivo recovery of irradiated red cells.

BACKGROUND: Transfusion-associated graft-versus-host disease can be prevented by gamma radiation of blood components. The increased use of blood components donated for patients by their family members has resulted in an increased demand for the storage and handling of irradiated units, and the ability to freeze the cells would allow storage beyond their current expiration date. STUDY DESIGN AND METHODS: To assess the effect of freezing and deglycerolization on irradiated red cells, studies of autologous radiolabeled red cell recovery were performed using normal volunteers. Each unit of CPDA-1 red cells was immediately divided into two equal volumes. Further handling of each half was identical except that one was irradiated (3500 cGy). The units were grouped under three protocols: I, irradiated on Day 0 and frozen on Day 5 (n = 4); II, irradiated on Day 7, rejuvenated, and frozen on Day 14 (n = 5); and III, irradiated on Day 14, rejuvenated, and frozen on Day 18 (n = 3). All cells were frozen for 3 to 10 months at -80 degrees C. RESULTS: Irradiated and control units showed no significant differences in supernatant potassium or hemoglobin. Autologous 24-hour posttransfusion recoveries (mean +/- SD) for the three groups were: I, 89.7 +/- 5.6 percent (control, 90.6 +/- 3.2%); II, 85.3 +/- 5.7 percent (control, 83.7 +/- 3.0%); and III, 79.5 +/- 1.4 percent (control, 82.6 +/- 5.2%). CONCLUSION: Irradiated red cells can be frozen after being stored under various conditions and can still meet established guidelines requiring 75-percent recovery 24 hours after transfusion.

Regulation of plasma factor XIII binding to fibrin in vitro.

The binding of plasma factor XIII to fibrinogen or fibrin that has been chemically or enzymatically induced to polymerize was studied. Factor XIII binding was assayed using a 3H-putrescine incorporation assay and an 125I-plasma factor XIII binding assay. More than 80% of the native and radiolabeled plasma factor XIII was bound to fibrin I formed by reptilase in EDTA, citrate, or heparin anticoagulated plasma. Plasma factor XIII and 125I-factor XIII was bound (89.6% to 92.5%) to fibrin II formed by thrombin in either citrate or EDTA anticoagulated plasma. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of 125I-plasma factor XIII bound to fibrin I or fibrin II formed by reptilase or thrombin in the presence of EDTA demonstrated the b2-subunit remained bound to the a-chains or thrombin-cleaved a-chains. In the presence of calcium chloride and thrombin, the b2-subunit dissociated and factor XIIIa was bound. Protamine sulfate caused fibrinogen polymerization in the absence of divalent cations and reduced both plasma factor XIII and immunologic fibrinogen levels. Fibrinogen polymerized by protamine sulfate bound plasma factor XIII and the a2-subunit of 125I-platelet factor XIII. Plasma factor XIII was also bound to sonicated non-cross-linked fibrin II in either normal plasma or afibrinogenemic plasma. Plasma levels of several coagulation proteins were unchanged after the addition of reptilase, protamine sulfate, or sonicated fibrin to plasma. These results demonstrate that a specific binding site for the a2-subunit of plasma factor XIII is present on polymerized fibrinogen, fibrin I, and fibrin II. Furthermore, the presence of divalent cations, thrombin-cleavage of plasma factor XIII, and release of fibrinopeptides A or B are not required for plasma factor XIII binding to polymerized fibrinogen and fibrin.

Antigens associated with the activation of murine mononuclear phagocytes in vivo: differential expression of lymphocyte function-associated antigen in the several stages of development.

Two well-characterized antigens [Mac-1 and lymphocyte-function-associated antigen (LFA-1)], expressed on a variety of leukocytes, are members of a family of surface proteins associated with multiple recognition functions. To analyze expression of these proteins during macrophage development, we utilized both radioimmunoassay and flow cytometry. As previously reported, Mac-1 is expressed on murine macrophages in all stages of development. We found LFA-1 to be present on murine mononuclear phagocytes but only in certain stages of their development. Specifically, we found LFA-1 was expressed on murine tissue macrophages but only on those activated in vivo by bacillus Calmette Guerin (BCG) or, to a lesser extent, primed by pyran copolymer. Although LFA-1 was absent on inflammatory (responsive) and resident tissue macrophages it was also present on blood-borne monocytes. Activated macrophages also selectively expressed the H-11 and Ly-6 antigens. Thus, these data indicate that LFA-1 is selectively expressed on mononuclear phagocytes of the tissues but only on those in the primed and activated stages of development.

The effect of fibrin polymers on thrombin-catalyzed plasma factor XIIIa formation.

The effect of fibrin polymers on thrombin-catalyzed factor XIIIa formation was studied in afibrinogenemic plasma. Fibrin polymers derived from des A fibrinogen and des A,B fibrinogen increased sixfold the rate of thrombin-catalyzed factor XIIIa formation in the presence of EDTA. Calcium chloride accelerated factor XIIIa formation 14-fold in the presence of des A,B fibrinogen without increasing the rate of thrombin formation. Fibrinopeptides A and B had no effect on factor XIIIa formation in afibrinogenemic plasma. Des A,B fibrinogen reduced by 20- to 40-fold the thrombin concentration required to activate factor XIII. Glycyl-L-prolyl-L-arginyl-L-proline (gly-pro-arg-pro), a fibrin polymerization inhibitor, inhibited des A and des A,B fibrinogen from enhancing thrombin-catalyzed factor XIIIa formation. Gly-pro-arg-pro did not modify factor XIIIa formation in afibrinogenemic plasma and did not inhibit thrombin cleavage of the chromogenic substrate S-2238. These results demonstrate that fibrin polymers accelerate thrombin-catalyzed plasma factor XIIIa formation.

Cleavage of blood coagulation factor XIII and fibrinogen by thrombin during in vitro clotting.

Thrombin cleavage of blood coagulation Factor XIII (a2b2) and fibrinogen was studied during in vitro clotting to determine the physiologic sequence of these events. First, the time course of fibrin formation and cleavage of Factor XIII was measured in platelet-rich plasma. Cleavage of fibrinogen was measured by using a radioimmunoassay for fibrinopeptide A. Conversion of trace amounts of radioiodinated a-chains of 125I-Factor XIII to thrombin-modified a-chains was measured in unreduced 10% sodium dodecyl sulfate-polyacrylamide gels. During spontaneous clotting, a similar percentage of 125I-Factor XIII and fibrinogen was cleaved at each time point. Visible gelation of polymerized fibrin monomer occurred when 24 +/- 8% of fibrinogen was cleaved and 21 +/- 6% of Factor XIII was converted to Factor XIII'. Thrombin cleavage of Factor XIII and fibrinogen was also studied in platelet-poor plasma to which thrombin was added. In order to measure Factor XIIIa activity, fibrin polymerization was completely inhibited by the addition of Gly-Pro-Arg-Pro. Factor XIIIa formation was measured by the incorporation of [3H]putrescine into casein. The concentration of added thrombin required to cleave 50% of fibrinogen and Factor XIII was 0.65 U/ml and 0.35 U/ml, respectively. The rate of cleavage of fibrinogen by thrombin was 43-fold greater than cleavage of Factor XIII. Lower Gly-Pro-Arg-Pro concentrations were used to determine the effects of incompletely inhibiting fibrin polymerization on cleavage of Factor XIII and fibrinogen. Thrombin cleavage of Factor XIII but not fibrinogen was dependent on the extent of fibrin polymerization. The more marked the degree of inhibition of fibrin polymerization, the slower the rate of Factor XIIIa formation. Thus, in platelet-rich plasma, thrombin cleavage of Factor XIII and fibrinogen are closely related events during spontaneous clotting. Furthermore, cleavage of Factor XIII during clotting is enhanced by fibrin polymerization in platelet-poor plasma.

Measurement of blood coagulation Factor XIIIa formation in plasma containing glycyl-L-prolyl-L-arginyl-L-proline.

A method to directly measure the formation of blood coagulation Factor XIIIa in platelet-poor plasma unmodified by heat is described. The synthetic peptide glycyl-L-prolyl-L-arginyl-L-proline, a fibrin-polymerization inhibitor, was used to prevent clotting of platelet-poor plasma. Plasma was diluted to a final concentration of 2.5% (v/v) in 0.1 M Tris-HCl, pH 8.5, buffer containing 25% glycerol, 5 mM calcium chloride, and 0.25 mM glycyl-L-prolyl-L-arginyl-L-proline and then activated by thrombin (20 U/ml) for 15 min. The Factor XIIIa-catalyzed incorporation of [3H]putrescine into Hammersten casein was used to measure Factor XIIIa formation. The assay detected Factor XIIIa in 2.5 to 50 microliter of thrombin-treated plasma. When purified Factor XIII was added to Factor XIII-deficient plasma, there was complete recovery of the Factor XIII added. Glycyl-L-prolyl-L-arginyl-L-proline did not inhibit Factor XIIIa activity in thrombin-treated plasma or purified platelet Factor XIIIa. Glycerol stabilized Factor XIIIa activity in thrombin-treated plasma and buffer for 60 min. The presence of fibrinogen in plasma did not modify the assay results. The time course of thrombin-catalyzed Factor XIIIa formation in platelet-poor plasma containing glycyl-L-prolyl-L-arginyl-L-proline was directly measured using the assay.