Joseph C. Fratantoni

Consequences of chemical modifications on the free radical reactions of human hemoglobin

Hemoglobin-based oxygen carriers (HBOCs) are candidates for use as blood substitutes and resuscitation fluids. We determined that HBOCs of specific types differ in their ability to generate or interact with free radicals. The differences do not correlate with oxygen affinity. Detailed comparisons with unmodified human hemoglobin, HbA0, were carried out with two cross-linked derivatives: HbA-FMDA, produced by the reaction of human oxyhemoglobin with fumaryl monodibromoaspirin, and HbA-DBBF, produced by the reaction of human deoxyhemoglobin with bis(3,5-dibromosalicyl) fumarate. Both derivatives had lower oxygen affinity than unmodified HbA0. As previously reported, exposure of oxyhemoglobin to H2O2 causes generation of free radicals capable of generating formaldehyde from dimethyl sulfoxide. Relative to the reaction catalyzed by 50 microM HbA (18.0 +/- 3.5 nmol/30 min/ml), the formaldehyde formation was roughly 70% for HbA-DBBF and 50% for HbA-FMDA under comparable conditions. More profound differences are exhibited at lower hemoglobin concentrations. Spectral changes of the HBOCs during the reaction differ qualitatively and occur at different rates. The HBOCs also differ in rates of hemoglobin-catalyzed NADPH oxidation and aniline hydroxylation, reactions mediated by reactive oxygen species. These results show that stereochemical differences brought about by chemical cross-linking alter the ability of HBOCs to generate radicals and to react with activated oxygen species. These studies also show that the ability of hemoglobin to produce activated species of oxygen can be enhanced or suppressed independently of oxygen affinity.

Frozen platelets and platelet substitutes in transfusion medicine

uring the past few years, efforts to improve platelet storage by cryopreservation and to develop platelet substitutes have been undertaken in multiple academic and commercial settings. A major goal for the researchers and regulators who work with these products is to establish methods for the assessment of hemostatic function in vitro, in animal models, and in clinical trials. To understand how platelet products can be developed for field medicine and to facilitate research in this area, the Combat Casualty Care Programs of the Naval Research and Development Command and the Medical Research and Materiel Command of the United States Army sponsored a conference called “Frozen Platelets and Platelet Products in Transfusion Medicine” on March 7 and 8, 1996, at the Uniformed Services University of the Health Sciences in Bethesda, MD. Co-organizers and participants were the Center for Biologics Evaluation and Research, the Food and Drug Administration (FDA), and the Blood Resources Program of the National Heart, Lung, and Blood Institute, National Institutes of Health.

Characterization of the platelet response to exogenous arachidonic acid

Abstract When aggregation and release are the end points, washed human platelets treated with a range of concentrations of arachidonic acid display a bell-shaped dose-response curve. The peak is at approximately 200 μM, followed by a sharp decrease with minimum reaction at 400 μM. Prostaglandin synthesis, measured by malondialdehyde production, reaches peak activity at 400–500 μM and does not decrease. Platelet lysis, as manifested by lactate dehydrogenase release, increases only in response to arachidonate concentrations above 400 μM. When platelets are treated with 400 μM arachidonate for three minutes, subsequent reaction with thrombin, but not with A23187, is inhibited. This inhibitory activity is not sensitive to aspirin, indomethacin or ETYA and cannot be transferred in the supernatant fraction of the platelet suspension. The inhibitory activity generated from platelets reacted with 400 μM arachidonate is not a product of the cyclooxygenase or lipoxygenase systems.

Aberrant morphology of platelets stored in five day containers.

Platelets for transfusion, stored in either of two new types of container (PL-732 and CLX), demonstrated unusual morphological alterations after 2 or 3 days of storage. The atypical forms observed included crescents, elongated tubular forms and rings. Development of these forms was not seen if the permeability of the container was inhibited and the pH kept below 6.7. The variables which differentiate these new containers from those previously used, and which appear to be related to the changes described, are pH, pO2 and presence of a leachable plasticizer. The functional behaviour of these platelets, as assessed by serotonin uptake and resistance to hypotonic shock, was not different from that of control platelets.

Ouabain affects platelet reactivity as measured in vitro

Ouabain, a digitalis glycoside and an inhibitor of the Mg2+-dependent Na+-K+ ATPase, was used to probe the role of intracellular Na+ levels in the regulation of platelet reactivity. Platelets preincubated with 10 to 150 microM ouabain exhibited a potentiated aggregation response to collagen (14.4 to 180 micrograms/mL), ADP (4 to 12 microM) and thrombin (0.03 to 0.10 unit/mL). Ouabain markedly decreased the time interval between addition of collagen and the onset of shape change. At submaximal concentrations of collagen, thrombin and ADP, preincubation with ouabain increased the rate and amplitude of the aggregation response. Irreversible aggregation was achieved in ouabain-treated platelets by using concentrations of ADP which induced only reversible aggregation in the absence of ouabain. In addition, chelation of extracellular calcium with EGTA or EDTA (2 mM) failed to block reactivity to collagen, ADP or thrombin in ouabain-treated platelets. These results suggest that ouabain induces a preactivation state in platelets, perhaps via modulation of intracellular Na+ levels.

Demonstration of the Efficacy of a Therapeutic Agent

The FDA is charged with ensuring that drugs and biologies are pure, potent, safe and effective. In this brief discussion, we will consider the regulatory basis for the demonstration of clinical efficacy required by FDA. We will also discuss the nature and variety of clinical trial end-points.

Dimethyl sulfoxide: effects on function of fresh platelets and on the viability of platelets in storage

Dimethyl sulfoxide (DMSO) is used as a cryoprotective agent when platelets are frozen. We examined the effect of DMSO (0.1 to 10%) on platelet aggregation, release, and prostaglandin synthesis (as indicated by malondialdehyde formation) in response to thrombin, collagen, arachidonic acid and calcium ionophore. Inhibition was observed at the lowest levels of DMSO, varied with the type of stimulus, and was reversed by washing the platelets. Inhibition of aggregation, release, and malondialdehyde formation were dose‐dependent with thrombin or collagen. DMSO did not inhibit malondialdehyde formation stimulated by arachidonic acid, nor did it consistently inhibit any function stimulated by calcium ionophore. When platelets were stored as platelet‐rich plasma at 20 to 24°C for 48 hours, with and without 5 percent DMSO, and subsequently washed, the platelets stored with DMSO were more reactive in vitro. These results indicate that platelet function inhibition by DMSO not only is reversible, but protects the platelets during storage. The factor limiting the use of DMSO in platelet storage is potential systemic toxicity, not its effects on platelets.

Oxidation reactions of human, opossum (Didelphis virginiana) and spot (Leiostomus xanthurus) hemoglobins: a search for a correlation with some structural-functional properties

1. Relative to human HbA, opossum (Didelphis virginiana) hemoglobin was found to be more susceptible to autoxidation. While the initial rate of autoxidation of spot (Leiostomus xanthurus) hemoglobin is close to that of HbA, complete oxidation occurs in 50 hr. 2. Direct addition of hydrogen peroxide (H2O2) induced oxidation of hemoglobins in a definite order: spot Hb > HbA > opossum Hb. Excess H2O2 led to heme degradation and precipitation that occurred much faster for spot Hb than the case with other proteins. 3. Exposure of hemoglobins to a continuous flux of H2O2, generated by the glucose/glucose oxidase system, induced the formation of heterogeneous protein-associated oxidation products. 4. Differential reactivity among these hemoglobins under the same or different oxidative conditions, with respect to methemoglobin formation and stability of the ferric form, may reflect the differences in the local heme environment of these proteins.

Hemoglobin-based oxygen carriers (HBOCs): structural alterations that affect free radical generation.

We examined how changes in oxygen affinity brought about by different chemical modifications of hemoglobins affect their oxidation-reduction reactions. The three modified hemoglobins studied were HbA-FMDA, HbBv-FMDA, produced by the reaction of human or bovine oxyHb with fumaryl mono-dibromoaspirin; and HbA-DBBF, produced by the reaction of human deoxyHb with bis(3,5-dibromosalicyl) fumarate. Exposure of oxyHb to H2O2 causes generation of free radicals capable of cleaving dimethylsulfoxide (Me2SO) to produce formaldehyde (HCHO). Relative to the reaction rate for HbAo (630 +/- 130 M/min) the rates of HCHO formation were roughly 70% for HbA-DBBF, 50% for HbA-FMDA and 16% for HbBv-FMDA. Exposure to H2O2 also caused spectral changes at varied rates for the HBOCs analyzed. Although these rates were not directly correlated with the rates of free radical formation, addition of mannitol or thiourea slowed both the rate of spectral changes and HCHO formation. The relative ability of the ferric derivatives of the HBOCs to participate in free radical reactions was monitored by assays of non-enzymatic NADPH oxidation and aniline hydroxylation. HbBv-FMDA showed significantly slower rates than the other HBOCs in both assays. The observed differences between HBOCs in these assays indicate differences in their ability to generate or interact with free radicals.

Which Are the Principal Established or Potential Risks for Donors Undergoing Cytapheresis Procedures and How Can They Be Prevented

H. Borberg. Side effects occurring during the utilization of blood cell separators may be related to the equipment or to simultaneously applied supporting agents. So far, there has been no reason to discuss whether any of the disposables in current use may lead to reactions like temporary decrease of leucocytes or complement activation described for other extracorporeal systems [1,2]. It may thus be assumed that side effects occurring during the application of blood cell separators are rather due to the technique of cytapheresis than to the equipment itself. This does not imply that undesirable reactions may not occur due to an inaccurate usage of the equipment. Intermittent flow centrifugation, for instance, applying disposable bowls, filled with cells rather than whole blood at the end of each cycle, may lead to a volume challenge, if the size of an appropriate bowl has not correctly been chosen. Also, by way of precaution, the operator may wish to control the donors blood pressure routinely at the end of each cycle, to maintain a proper control of the circulation, especially in labile donors. Platelet collection is generally performed using 2-3% citrate in a ratio of 1:8-1:12 for anticoagulation. An even lower concentration of about 1.5% may in rare instances lead to platelet aggregation, whereas hypocalcaemia occurs if a high citrate concentration, which does not meet the requirements of the donor, is used. Since we cannot pro-