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Biomedical subjects

A P Bode

Publications and source records attributed to A P Bode.

34 records · Page 2Linked to original sources
PubMed1990

Platelet activation may explain the storage lesion in platelet concentrates.

While the exact nature of the dysfunction of stored platelets is not known, it is generally agreed that the platelet's metabolic activity with lactate accumulation presents a significant impediment to prolonged storage. There is an increasing body of evidence that stored platelets have become activated in the preparation and handling of platelet concentrates. Changes in platelet function and structure in concentrates can be explained in terms of sequelae of activation, especially heightened metabolic activity and activation-specific changes in surface glycoproteins on stored platelets. With the use of inhibitors of platelet activation in the preparation of platelet concentrates, the loss of platelet function and integrity is less rapid and platelet metabolic rate is decreased during an extended storage period. Surface levels of glycoprotein Ib, normally decreased during prolonged storage of platelets, are well-preserved in the presence of activation inhibitors. When the use of inhibitors is combined with replacement of plasma with an artificial medium, platelets stored for up to 20 days appear to be metabolically and structurally intact and responsive to stimuli. In summary, platelet activation appears to play a major role in the generation of the storage lesion in platelet concentrates.

Blood Preservation↗
PubMed1990

Effect of platelet activation inhibitors on the loss of glycoprotein Ib during storage of platelet concentrates.

Platelet membrane glycoprotein Ib (GPIb) in stored platelet concentrates was analyzed by flow cytometry with three separate monoclonal antibodies (AN-51, 6D1, and SZ-2), by tritiated glycoprotein radiolabeling, and by ristocetin-induced agglutination. Flow cytometry showed that a population of surface GPIb-negative platelets was evident at 5 days and increased three- to fivefold by the tenth day. Tritium radiolabeling of surface GPIb showed a decrease over 10 days of 37% +/- 17%. The degree of loss of surface GPIb correlated well with other changes during storage: decreased ristocetin-induced agglutination, decreased responsiveness in the hypotonic shock test, lower plasma pH, and increased extracellular lactic dehydrogenase. Immunoblotting of total platelet GPIb with the SZ-2 antibody showed a decrease of 58% +/- 16% during the 10-day storage period. The effect of protease inhibitors or platelet activation inhibitors on the loss of GPIb during storage was studied in platelet concentrates paired with untreated controls. Only the platelet activation inhibitors prostaglandin E1 and theophylline retarded the loss of surface GPIb levels (93% +/- 5% GPIb remaining vs 65% +/- 16%). Total GPIb levels also decreased less in the presence of the activation inhibitors (45% +/- 22% lost vs 70% +/- 14% lost). These findings suggest that platelet activation, rather than plasma enzymatic activity, is responsible for the loss of platelet GPIb during storage of platelet concentrates.

Alprostadil↗
PubMed1989

Lysed platelets shorten the activated coagulation time (ACT) of heparinized blood.

Aliquots of heparinized whole blood were incubated at 37 degrees C with preparations of lysed platelets or buffer. In the presence of lysed platelets, the ACT decreased to 127 seconds (+/- 41, n = 9) versus 248 seconds (+/- 43, n = 9) in the controls (P = 0.0002). The shortening of the ACT by lysed platelets followed a typical dose-response relationship. Fractionation of lysed platelet preparations revealed that the procoagulant membrane fraction, not the cytosol fraction containing PF4, was responsible for this effect of lysed platelets on the ACT. These findings demonstrate that the ACT can be artifactually low in the presence of heparin, indicating a potential problem in the use of the ACT for monitoring of heparin neutralization.

Blood Coagulation Tests↗
PubMed1989

Metabolic status of platelet concentrates during extended storage: improvement with pharmacological inhibitors and reduced surface-to-volume ratio.

The depletion of plasma nutrients and buffering capacity may present a potential barrier to the long-term liquid storage of platelet concentrates (PC). We have found that PC prepared with reversible inhibitors of platelet activation added to the citrate anticoagulant and stored at a reduced surface-to-volume (S/V) ratio have a much slower rate of lactate build-up (p less than 0.01), slower consumption of glucose (p = 0.05), and more stable pH (p less than 0.01) than controls. By pO2 and pCO2 measurements, PC prepared with inhibitors showed evidence of continued respiration and responsiveness even after storage at 22 degrees C for 15 days. In addition, these PC released only 11% of the total cellular LDH during the storage period as compared to the release of 43-67% of the total LDH in control PC. Maximum benefit of the inhibitors was seen after reduction of the S/V ratio of the storage container, which was made possible by the reduced metabolic demands of platelets stored in the unactivated state. These data suggest that the fall in pH and loss of platelet integrity associated with the platelet storage lesion are correlated with a high metabolic rate which can be controlled by inhibiting the activation of platelets during preparation and storage. The use of these inhibitors and reduced bag surface area may make prolonged liquid storage of platelets feasible.

Alprostadil↗
PubMed1989

The use of thrombin inhibitors and aprotinin in the preservation of platelets stored for transfusion.

We have evaluated the use of several thrombin inhibitors (heparin, Fragmin, hirudin, and Thromstop) in combination with cyclic adenosine 3',5'-monophosphate-active agents (prostaglandin E-1 [PGE-1] plus theophylline) and aprotinin for preparation and extended storage of platelet concentrates. Replacement of citrate with heparin resulted in accelerated clumping of platelets during storage. Fragmin, a low molecular weight heparin fraction, induced a high degree of platelet clumping, even in the presence of a standard amount of citrate (citrate-phosphate-dextrose-adenine formula 1 [CPDA-1]) plus PGE-1 and theophylline. The best anticoagulant formulation appeared to be CPDA-1 containing PGE-1 plus theophylline plus aprotinin, plus either hirudin or Thromstop for preservation of platelet responsiveness and structural integrity by in vitro markers. In eight platelet concentrates prepared with these inhibitors and stored for 15 days, the plasma pH was 6.50 +/- 0.15, the PO2 was 94 +/- 29 mm Hg, the PCO2 was 27 +/- 4 mm Hg, and the hypotonic shock response was 76% +/- 25% of the initial value recorded at Day 1 (all values expressed as mean +/- SD). Only 11% +/- 3% of the cellular lactate dehydrogenase was released, 69% +/- 8% of the platelets appeared to be in a discoid shape, and the response to 20 mumol/L adenosine 5'-diphosphate remained at 50% +/- 17% of the initial value. These results were all significantly different (p less than 0.01) from data obtained for concentrates prepared without aprotinin. The use of a factor Xa inhibitor (diamidino-benzofuranyl ethane) in place of Thromstop or hirudin did not provide substantial improvement over controls without inhibitors.

Aprotinin↗
PubMed1989

Plasmin activity and complement activation during storage of citrated platelet concentrates.

Platelet concentrates were studied for evidence of plasmin activity and complement activation during a 7-to-10-day storage period. When measured by an amidolytic activity assay, plasmin reached a level of 845 +/- 540 nkats/L on day 7 (n = 9). Fibrin(ogen) degradation product (FDP) levels became markedly elevated on the tenth day of storage, rising to 45 +/- 22 micrograms/ml (n = 5). Antiplasmin levels decreased in platelet concentrates by 18% +/- 6% (n = 5) over 7 days, but there was no significant decrease in stored platelet-poor plasma (-1.7%, n = 5, p = 0.5). The amount of plasminogen in platelet concentrate converted to plasmin was estimated to be less than 3% by assay of total plasminogen. Supernatant plasma from stored platelet concentrates was examined for the presence of the complement activation peptides C3a and C5a. From day 0 to day 10 of storage, mean C3a levels rose from 327 ng/ml to 6690 ng/ml. An equivalent increase in C3a levels, from 336 ng/ml at day 0 to 6866 ng/ml at day 10, was also observed in stored platelet-poor plasma. C5a was not detected (less than 10 ng/ml) at any point during the storage period; however, we noted a small decrease of borderline significance (p = 0.04) in total C5 from day 0 (117 micrograms/ml) to day 10 (108 micrograms/ml). Only trace amounts of C3 fragments were found on stored platelets, and there was no evidence of the membrane attack complex. These findings indicate the presence of plasmin activity and conversion of C3 during storage of platelet concentrates.

Blood Platelets↗
PubMed1988

Preservation of in vitro function of platelets stored in the presence of inhibitors of platelet activation and a specific inhibitor of thrombin.

We prepared platelet concentrates (PCs) in an experimental anticoagulant consisting of citrate-phosphate-dextrose-adenine formula 1 (CPDA-1) containing inhibitors of platelet activation (prostaglandin E1 and theophylline) and a specific thrombin inhibitor (hirudin or D-phenylalanyl-L-prolyl-L-arginine-chloromethyl ketone-2HCl). Over a 15-day storage period, these PCs showed by in vitro markers (pH, lactic dehydrogenase, PO2, PCO2, glucose consumption, morphology, and hypotonic shock recovery) greatly improved function and integrity relative to PCs in CPDA-1 only. At day 10 of storage, the experimental PCs had higher pH (P = 0.02), better morphology scores (P less than 0.001), more rapid recovery from hypotonic shock (P = 0.03), and higher PCO2 levels (P less than 0.001) than controls in CPDA-1 only. Additional storage time increased these differences to a significance level of p less than 0.01 in all tests. The experimental anticoagulant was less effective when hirudin was absent. Plasma pH was well maintained, and less glucose was consumed in the experimental anticoagulant without the aid of an increase of buffering capacity or gas transport. These findings indicate that thrombin and platelet activation play a major role in the development of the platelet storage lesion.

Adenine↗
PubMed1986

Generation and degradation of fibrinopeptide A in stored platelet concentrate.

The levels of fibrinopeptide A (FPA) were measured in samples from stored platelet concentrates (PC) by radioimmunoassay. In 27 standard, citrated PC, the mean FPA was 13.6 ng/ml, which is elevated 5-6X over background levels. This value did not change significantly over a 7-day storage period. Addition of PGE-1 and theophylline resulted in higher initial levels of FPA (18.0 ng/ml) and a pronounced rise during the storage period (to 43.4 ng/ml by day 10). In contrast, addition of a thrombin inhibitor, D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone-2 HCl (PPACK) or hirudin, resulted in lower initial levels of FPA relative to standard, citrated PC and a slow increase over time. Introduction of exogenous FPA into citrated PC resulted in a predicted elevation of FPA levels followed by a rapid loss of immunoreactivity (t1/2 = 18 h). Addition of PPACK did not affect this fall-off. However, PC prepared and stored in the presence of PGE-1 and theophylline showed a much slower fall-off of exogenous FPA (t1/2 = 38 h). These data indicate that FPA levels in samples from citrated PC represent a dynamic balance between generation and degradation processes and, thus, the data above underestimate the amount of thrombin activity present in stored, citrated PC.

Amino Acid Chloromethyl Ketones↗
PubMed1986

Analysis of platelet factor 3 in platelet concentrates stored for transfusion.

The amount of platelet factor 3 (PF3) activity expressed in stored platelet concentrates (PC) was measured in conjunction with extracellular LDH levels. Standard manual techniques for preparation of PC resulted in PF3 and LDH levels remarkably higher than those observed in PC prepared by apheresis or special manual plateletpheresis. During storage of PC, PF3 activity rose 2- to 10-fold, while LDH levels rose less than 2-fold over starting values. Loss of LDH and appearance of PF3 expressed as a percent of total per platelet were significantly correlated only in standard, manual PC. Approximately half of the PF3 activity observed in any type of PC remained in the supernatant plasma after centrifugation. Upon gel filtration, the supernatant PF3 activity eluted in a high molecular weight peak containing phosphate and light-scattering material. Our findings indicate that platelets in standard, citrated PC express PF3 in amounts that approach that of frozen-thawed (lysed) platelets; however, the manner in which the PF3 activity appears suggests that stored platelets undergo a combination of activation and damage processes.

Blood Coagulation Factors↗
PubMed1985

Association of factor V activity with membranous vesicles released from human platelets: requirement for platelet stimulation.

The membrane-associated factor V-like activity (platelet factor 1, PF1) and the phospholipid-like catalytic surface activity (platelet factor 3, PF3) were studied in human platelets from normal and two factor V-deficient donors. Collagen stimulation or mechanical disruption of gel-filtered platelets was necessary for the expression of significant amounts of PF1 and PF3. Stimulation was also necessary for the uptake of factor V or Va by PF1-deficient platelets from the factor V-deficient donors. The activity of PF1 was also generated by association of factor V or Va with membrane-rich fractions obtained by gel filtration of the supernatant from collagen-stimulated or frozen-thawed PF1-deficient platelets. The amount of PF1 obtained by such all-or-none binding experiments was directly proportional to the amount of PF3 already expressed in the platelet preparation. These data have been summarized in terms of a hypothesis which views PF1 and PF3 to be activities associated with membranous vesicles released from platelets only after stimulation.

Blood Coagulation Factors↗
PubMed1985

Expression of coagulant activity in human platelets: release of membranous vesicles providing platelet factor 1 and platelet factor 3.

The relationship between the appearance of membrane-associated factor V-like activity (platelet factor 1, PF1) and phospholipid-like catalytic activity (platelet factor 3, PF3) has been examined, in vitro, in collagen-stimulated, human platelets. Both activities increased 7 fold upon collagen treatment relative to stirred controls. After sedimentation of stimulated platelets, 31% of total PF1 and 41% of PF3 remained in the supernatant fraction. PF1 eluted from a Sepharose CL-4B column in the same void volume fractions as PF3, phospholipid, and vesicular particles. These fractions had roughly 100 fold (lipid basis) or 1000 fold (protein basis) enhanced specific activity when compared to the stimulated platelet suspension. Freeze-fracture electron microscopy demonstrated that these void volume fractions contain two populations of membranous vesicles (80-200 nm and 400-600 nm in diameter). Upon centrifugation of the void volume fractions, PF1 and PF3 activities, phosphate-containing material, and ultraviolet-absorbing material all sedimented at the same rate, indicating that PF1 and PF3 are activities associated with one or both of the platelet-derived vesicle populations. Finally, we examined the effects of inhibitors on the appearance of PF1, PF3, platelet factor 4, total intrinsic factor V activity, and serotonin as well as on platelet aggregation. These studies suggest that the collagen-stimulated release of PF1 and PF3 is not coupled to either platelet aggregation or PF4 release but is probably a separate phase of the release reaction.

Blood Coagulation Factors↗
PubMedDate not supplied

Lyophilized platelets: continued development.

In our initial investigation of functionality of platelets freeze-dried after stabilization with 1.8% paraformaldehyde, we found that the rehydrated cells were morphologically intact and retained adhesive and procoagulant properties. Further testing of fixed, washed freeze-dried platelets has demonstrated the physiologic nature of their adhesion in vitro and their hemostatic efficacy in vivo in correcting the bleeding time in thrombocytopenic animal models. Binding studies with monoclonal antibodies and radiolabelled ligands indicate an intact GpIb vonWillebrands factor receptor as on fresh platelets, but a somewhat attenuated GpIIbIIIa fibrinogen receptor. Repeated infusion of canine lyophilized platelet preparations in a single recipient over several months has shown no incipient cytopenia upon infusion of new doses nor accelerated clearance of platelets. These findings suggest minimal risk of systemic thrombosis or severe immunogenic reaction and support the notion of approaching clinical trials as soon as possible.

Animals↗
PubMedDate not supplied

Plasma levels of ionized and total calcium during storage of citrated platelet concentrate.

Measurements of ionized and total calcium levels in supernatant plasma samples from citrated platelet concentrates (PCs) were made over 7 days of storage. Both ionized and total calcium increased significantly during the storage period: respectively, from 0.074 mM Ca2+ in fresh platelet-rich plasma to 0.084 mM in PCs stored for 7 days (p = 0.017), and from 1.94 mM total calcium to 2.06 mM (p = 0.014) over the same period. The increase in calcium was partially blocked by the addition of platelet activation inhibitors to the PCs. Platelet-poor plasma stored under similar conditions showed no significant change in ionized or total calcium, which indicated that the increases observed in PCs were due to the release of cellular calcium. Significant correlations (p less than 0.01) were found between ionized or total calcium levels and lactate concentration or pH, but not hypotonic shock recovery rate. The demonstration of non-zero levels of ionized calcium makes it likely that Ca2+-dependent enzyme systems such as calpain expression and thrombin generation are active in the plasma of citrated PCs and may contribute to the platelet storage lesion.

Alprostadil↗
PubMedDate not supplied

In-line leukocyte filtration during bypass. Clinical results from a randomized prospective trial.

Leukocyte mediated pulmonary injury may delay recovery after cardiac surgery, and leukocyte depletion during bypass has been suggested. Two groups of patients were randomly, prospectively assigned from 50 sequential patients to undergo open heart surgery using cardiopulmonary bypass, either with (n = 25) or without (n = 25) leukocyte filters. The two groups were not significantly different regarding age, gender, race, pre-operative ejection fraction, pump time, or cross-clamp time. Post operative arterial blood gases (pO2: 173 +/- 66 vs 192 +/- 107; pCO2: 30.2 +/- 8.2 vs 30.8 +/- 8.0), pulmonary vascular resistance (PVR 105 +/- 45 vs 112 +/- 50 dyne cm-5), time on ventilator (17.8 +/- 6.4 vs 19.7 +/- 8.6 hr), and length of hospital stay (7.65 +/- 4.57 vs 8.52 +/- 5.87 days) were not different between groups (mean +/- SD, with vs without filters, respectively). Arterial oxygenation was somewhat poorer, and PVR was somewhat lower in the leukocyte filtered group. However, these trends did not produce significant decreases in total ventilator time or length of hospital stay. In-line filtration did remove leukocytes, but did not reduce circulating leukocyte count. In effect, leukocyte filtration produced an effective leukocyte concentration at the filter site. These data do not support routine incorporation of in-line leukocyte filtration during bypass.

Cardiac Surgical Procedures↗