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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., (Copyright 1999 Academic Press.)

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.

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.

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 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.