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Biomedical subjects

V P Addonizio

Publications and source records attributed to V P Addonizio.

At least 55 records · Page 3Linked to original sources
PubMed1985

Iloprost (ZK36374), a stable analogue of prostacyclin, preserves platelets during simulated extracorporeal circulation.

Contact between blood and synthetic surfaces of an extracorporeal circuit results in extensive alterations in platelet function. These platelet abnormalities have been reproduced in vitro by recirculating heparinized (5 units/ml) human blood at 37 degrees C in a silicone rubber circuit (0.1 m2) containing a spiral coil membrane oxygenator (0.9 m2). During control recirculation trials, platelet counts fell to 29% +/- 8% (mean +/- standard error of the mean) of initial levels within 30 minutes, and sensitivity to the soluble agonists, adenosine diphosphate and epinephrine, disappeared. Plasma levels of the platelet-specific protein platelet factor 4 rose to 2,600 +/- 200 ng/ml, indicating extensive platelet granule release. Similarly, plasma concentrations of thromboxane B2 rose from less than or equal to 100 pg/ml to 500 +/- 200 pg/ml at 120 minutes, and transmission electron microscopy demonstrated disruption of platelet subcellular architecture. In contrast, when ilioprost, a stable prostacyclin derivative (1 ng/ml), was added to the circuit prior to recirculation, the thrombocyte count remained at 85% +/- 4% of initial values. Platelets incubated or recirculated for 2 hours in the presence of iloprost responded normally to both adenosine diphosphate and epinephrine after separation from the drug by gel-filtration. Furthermore, plasma concentrations of platelet factor 4 remained below 300 ng/ml, plasma levels of thromboxane B2 failed to reach detectable levels, and platelet ultrastructure remained intact. Thus, iloprost effectively preserves the circulating platelet count, prevents platelet granule release, and preserves platelet functional and morphological integrity during simulated extracorporeal circulation.

Blood Platelets↗
PubMed1982

Inhibition of human platelet function by verapamil.

We evaluated the ability of the calcium antagonist, verapamil, to alter human platelet function. With the concentrations tested (20 ng to 0.1 mg/ml of whole blood), verapamil inhibited epinephrine-induced aggregation and release of 14C-serotonin; produced a dose-dependent inhibition of 14C-serotonin uptake and prevented aggregation dependent release of thromboxane B2. The action of verapamil could be overcome by higher concentrations of both epinephrine and calcium. Furthermore, verapamil-induced inhibition could be reversed by gel-filtering platelets suggesting that verapamil's anti-platelet activity does not outlast its presence in plasma. Verapamil was relatively ineffective as an inhibitor of ADP-induced aggregation. As with epinephrine-induced platelet activation, the effects of verapamil on ADP-induced 14C-serotonin and thromboxane release correlated with its effects on secondary aggregation. Finally, verapamil failed to alter calcium ionophore-induced platelet aggregation. Thus, verapamil at the concentrations tested, appears to be functioning as a reversible, relatively specific inhibitor of epinephrine-induced platelet activation. Our findings suggest that the actions of verapamil in this regard are complex; there may be competitive inhibition of epinephrine binding as well as a blockade of epinephrine-induced calcium flux.

Blood Platelets↗
PubMed1982

Platelet-mediated cardiac ischemia.

Although platelets have been associated with angina pectoris, myocardial infarction, and sudden death, the platelet's capacity for induction and propagation of cardiac ischemia remains incompletely defined. We therefore evaluated the effects of platelet activation occurring within the coronary circulation and tested the hypothesis that inhibition of platelet function would prevent platelet-induced cardiac ischemia. Human platelets were isolated from blood obtained from normal donors by Sepharose 2B column chromatography, resuspended in Hepes buffer, and added to the perfusate of a Langendorff rabbit heart (platelet counts greater than 10,000/microliters). Without, and with low dose (10 microM) prostaglandin E1 (PGE1), a reversible inhibitor of platelet function, immediate and irreversible global cardiac ischemia, as monitored by NADH fluorescent photography, ensued (N = 4) following platelet activation with thrombin (0.1 to 1 U/ml). Higher concentrations of PGE1 (0.1 to 1 mM, N = 2) or aspirin ingestion (1000 mg taken approximately 12, 4, and 1 hr prior to experiment, N = 2) completely prevented this platelet-induced myocardial ischemia. Aspirin, unlike PGE1, was effective despite its inability to block thrombin-induced platelet aggregation in our in vitro gel-filtered system. We conclude that activation of platelets within the coronary circulation is sufficient for induction of irreversible cardiac ischemia. The efficacy of aspirin, a cyclooxygenase inhibitor, further suggests that the products of arachidonate metabolism (e.g., thromboxanes) have a fundamental role in the genesis of platelet-mediated myocardial ischemia.

Animals↗
PubMed1982

Platelets and extracorporeal circulation.

Extensive contact between blood and the synthetic surfaces of an extra-corporeal circuit causes thrombocytopenia, release of platelet granular contents, initiation of thromboxane synthesis, disruption of subcellular architecture and loss of platelet sensitivity to standard platelet agonists. All too frequently, these adverse platelet alterations are reflected in a prolongation of the post-operative bleeding time and excessive blood loss which precludes implementation of long-term circulatory assist devices. Unfortunately, a truly biocompatible material does not exist and efficiency of gas transport demands haemodynamic designs which actually promote platelet injury. Although manipulation of surface properties and mechanical improvements in circuitry have managed to reduce platelet-surface interactions, the ultimate potential of these manoeuvres may be limited. Synthetic surfaces and soluble agonists, however, appear to modulate similar pathways suggesting that temporary platelet inhibition might provide significant protection by preserving the morphological and functional integrity of circulating platelets during contact with extracorporeal circuits.

Blood Platelets↗
PubMed1982

Interaction of verapamil with human platelet alpha-adrenergic receptors.

Platelet aggregation induced by epinephrine is an alpha-adrenergic event and is inhibited in vitro by the calcium-channel blocker, verapamil. We wondered whether this inhibition might be mediated by an interaction of verapamil with platelet alpha-adrenergic receptors. Verapamil inhibited the specific binding of [3H]dihydroergocryptine ([3H]DHE), a nonselective alpha-adrenergic antagonist, to intact platelets in a concentration-dependent and competitive manner. At 10 microM verapamil caused a fivefold decrease in the affinity of binding of [3H]DHE to platelet alpha-adrenergic receptors (P less than 0.001) with no change in the number of receptors per platelet. The inhibition of binding was not reversed by excess calcium. Verapamil also inhibited the binding of a selective alpha 2-adrenergic antagonist, [3H]yohimbine, to intact platelets, and inhibited the binding of an alpha 2-partial agonist, [3H]clonidine, to platelet membranes. Moreover there was a strong correlation between verapamil's effect on [3H]DHE binding and its effect on epinephrine-induced aggregation (r = 0.98). Although verapamil is commonly used as an inhibitor of calcium flux across cell membranes, the present studies demonstrate that verapamil also inhibits epinephrine-induced aggregation at the level of the platelet alpha-adrenergic receptor.

Binding, Competitive↗
PubMed1982

Assay of prekallikrein in human plasma: comparison of amidolytic, esterolytic, coagulation, and immunochemical assays.

Using the substrate H-D-Pro-Phe-Arg-p-nitroanilide-HCl, an amidolytic assay was designed to measure prekallikrein in plasma. At a substrate concentration of 1 mM (Km = 0.2 mM), the amidolysis of purified kallikrein at 1 coagulant unit/ml was observed to be 2.47 mumole/min/ml. Conditions for plasma prekallikrein activation were optimized to approach complete activation when compared to the amidolytic activity of the purified plasma kallikrein. Plasma treated with chloroform to destroy inhibitors of kallikrein was activated with dilute kaolin (final concentration 1 mg/ml) for 1 min at 25 degrees C. Activated plasma prekallikrein had 78% (1.92 mumole/min/ml) of activity of purified kallikrein at plasma concentration. Comparison of this amidolytic assay with immunochemical, esterolytic, and coagulant assays of three subject populations (normals, women on birth control pills, and patients with hepatocellular disease) showed good correlation both in normals and in the patient groups between the amidolytic and esterolytic assays (r = 0.89). Each enzymatic assay correlated with the immunochemical assay (r = 0.72, r = 0.68, respectively). However, comparison of each of these assays with the coagulant assay showed no significant correlation due to the large inherent error of the latter assay. This standardized plasma prekallikrein amidolytic assay should facilitate studies of plasma prekallikrein concentration in physiologic and pathologic conditions and help identify activation of the contact phase of coagulation in disease states.

Blood Coagulation Tests↗
PubMed1982

Release of lysosomal hydrolases during simulated extracorporeal circulation.

Contact with surfaces results in activation of formed blood elements. This study demonstrates that during extracorporeal circulation, release of lysosomal enzymes occurs concomitantly with platelet granule secretion. Fresh, heparinized human blood was recirculated for 2 hours at 1,000 ml/min at 37 degrees C in silicone rubber circuits containing a membrane oxygenator (0.85 m2). The plasma levels of a platelet-specific protein, low affinity platelet factor 4 (LA-PF4), rose from less than 0.5 to 16 +/- 3 SEM microgram/ml plasma, indicating extensive release of platelet alpha granule contents. Concurrently, plasma activity of acid phosphatase and N-acetyl-beta-glucosaminidase increased nearly fivefold. Platelet inhibition prevented release of LA-PF4 and reduced acid phosphatase levels, but failed to alter the levels of N-acetyl-beta-glucosaminidase. Lidocaine (10 to 20 microgram/ml), however, prevented the rise in N-acetyl-beta-glucosaminidase activity and diminished acid phosphatase levels without altering secretion of LA-PF4. The failure of platelet inhibitors to block the increase in N-acetyl-beta-glucosaminidase, and the efficacy of lidocaine to do so without altering LA-PF4 secretion, suggest that leukocytes, not platelets, are the primary source of this lysosomal enzyme. Although acid phosphatase activity measured in plasma my be derived from leukocytes, platelet membranes, and not lysosomes, appear to be a more likely source. Release of hydrolytic enzymes from formed blood elements during cardiopulmonary bypass may cause endothelial cell injury and thus contribute to the increased vascular permeability associated with extracorporeal circulation.

Acetylglucosaminidase↗
PubMed1982

Mediation of cardiac ischemia by thromboxanes released from human platelets.

We evaluated the consequences of platelet activation within the coronary circulation and determined the contribution of released thromboxanes, the most potent vasoconstrictors known, to the ensuing cardiac ischemia. Human platelets were isolated by sepharose column chromatography from blood of normal donors and added to the crystalloid perfusate of a Langendorff rabbit heart (platelet counts greater than or equal to 10,000/microliters). Following thrombin-induced (1 U/ml) platelet activation, the coronary flow decreased by 30 +/- 10% (mean +/- SEM, P less than 0.02), the mean concentration of thromboxane B2 in the coronary sinus effluent rose to 62 +/- 25 pmol/ml, and immediate, often irreversible cardiac ischemia as monitored by nicotinamide adenine dehydrogenase autofluorescence photography, ensued. However, with high concentrations of the platelet inhibitor and vasodilator, prostaglandin E1 (1.0 mM), the coronary flow increased by 50 +/-= 15%, and the epicardial fluorescence remained unchanged despite a small (10 +/- 3 pmol/ml) increase in coronary sinus thromboxanes. Platelets isolated from donors who ingested aspirin were incapable of thromboxane synthesis (less than 5 pmol/ml) but remained normally responsive to thrombin-induced activation. When these platelets were challenged by thrombin during cardiac perfusion, however, coronary flow and epicardial fluorescence remained unchanged. We conclude that platelet activation within the coronary circulation can induce irreversible cardiac ischemia, which, however, can be prevented by appropriate pharmacologic inhibition of platelet function. Furthermore, the fact that cardiac perfusion was preserved during a thrombin challenge of platelets from aspirin-treated donors establishes a fundamental role for the products of cyclooxygenase activity (e.g., thromboxanes) in the genesis of this form of myocardial ischemia.

Aspirin↗
PubMed1982

Platelet function during cardiac operation: comparison of membrane and bubble oxygenators.

The effects of cardiopulmonary bypass with bubble and membrane oxygenator systems on platelet function were studied in 26 patients who had elective coronary arterial bypass grafts. Fourteen patients were perfused with spiral coil membrane oxygenator systems, 12 with bubble oxygenator systems. During and after bypass, platelet counts decreased in both groups; however, when corrected for dilution, platelet counts did not change significantly in patients perfused with membrane oxygenators and increased slightly but significantly in those perfused with membrane oxygenators and increased slightly but significantly in those perfused with bubble oxygenator systems. During and 1 hour after bypass, the concentration of adenosine diphosphate (ADP) required to cause complete aggregation increased in both groups. Plasma low affinity platelet factor 4 (LA-PF4) increased significantly during and after bypass in both groups. However, the concentration of platelet adenine nucleotides and LA-PF4, measured only in patients perfused with membrane oxygenator systems, did not change. Bleeding times increased postoperatively in both groups and 18 hour blood losses were similar. Cardiopulmonary bypass with membrane and bubble oxygenator systems causes qualitatively similar losses in sensitivity to ADP and similar increases in bleeding times. The mechanism by which platelets are altered during cardiopulmonary bypass in obscure but is not due to partial depletion of granule contents in patients perfused with membrane oxygenators.

Adenosine Diphosphate↗
PubMed1981

Prostacyclin in lieu of anticoagulation with heparin for extracorporeal circulation.

Prolonged extracorporeal circulation (ECC) using heparin as anticoagulant may be associated with pronounced thrombocytopenia and excessive bleeding. We, therefore, tested the hypothesis that reversible inhibition of platelet function, in lieu of heparinization, might preserve platelets and prevent coagulation in a perfusion circuit. When 500 ml of fresh heparinized (one U/ml) human blood was recirculated in a perfusion circuit constructed of standard silicone rubber components and a membrane oxygenator (0.95 M2), platelet counts declined to 9 +/- 2 (SEM) % of initial levels within 15 mins; plasma levels of the platelet specific protein LA-PF4 rose to 15 +/- 2 micrograms/ml within one hour indicating extensive release of platelet granule contents, and leukocyte counts declined to 91 +/- 4% within 15 mins. Prostacyclin (PGI2, greater than or equal to 25 eta M) or prostaglandin E1 (20 microM) and theophylline (12 mM) prevented platelet loss and release of granule contents. When heparin was reversed with protamine, however, immediate coagulation ensured. This occurred despite the absence of detectable activation of Hageman factor as evidenced by stability of plasma concentrations of prekallikrein in systems anticoagulated with heparin or citrate and despite our inability to detect thromboplastin-like properties in isolated leukocytes. Thus, coagulation in the presence of platelet inhibition suggests that alternative pathways, independent of platelet activation may exist. Platelet inhibition does preserve platelets preventing contact initiated release, but cannot serve by itself for anticoagulation.

Blood Coagulation↗
PubMed1980

In vivo release and turnover of secreted platelet antiheparin proteins in rhesus monkey (Macaca mulatta).

Human and rhesus monkey platelets secrete at least two antiheparin proteins: platelet factor 4 (PF4) and low affinity platelet factor 4 (LA-PF4). Neither of these proteins showed species-related antigenic differences. As determined by radioimmunoassay, the levels of PF4 and LA-PF4 antigen per 10(9) monkey platelets amounted to 10.7 and 20.3 microgram, respectively. One milliliter of monkey plasma prepared from blood collected into an anticoagulant composed of EDTA, prostaglandin E1, and theophylline solution contained 22.4 ng LA-PF4 and 8.0 ng PF4. Concentrations of these two platelet-specific proteins in monkeys closely resembled levels found in human platelets and plasma. Infusion of prostacyclin (PGI2) (100 or 300 ng/kg/min) into monkeys for 15 min resulted in a significant decrease of plasma levels of LA-PF4 antigen and of PF4 by 40%--60% (p < 0.0001). This decrease was related to the inhibitory effect of PGI2 on the secretion of platelets stimulated by a catheter or by venipuncture. Longer infusion of PGI2 did not produce further significant change. The supernate obtained after aggregation of human platelets stimulated by thrombin was injected into monkeys receiving PGI2 infusion. The disappearance of LA-PF4 antigen in monkey plasma followed a biphasic exponential curve with half-lives for the fast and slow components of 8.4 and 63 min. PF4 disappeared faster but followed the same pattern (half-lives for the fast and slow component of 2.1 and 70 min). Analysis of the experimental data suggests that the low levels of secreted platelet proteins in monkey plasma are related to their minimal in vivo release and to their rapid clearance.

Animals↗
PubMed1979

Effects of prostaglandin E1 on platelet loss during in vivo and in vitro extracorporeal circulation with a bubble oxygenator.

Temporary inhibition of platelet function with prostaglandin E1 (PGE1) prevents platelets loss and functional alterations during extracorporeal circulation with a membrane oxygenator. This study evaluated the ability of PGE1 to prevent platelet injury in circuits containing a bubble oxygenator. During in vitro recirculation of human blood, the circulating platelet count, expressed as a percent of initial levels, decreased to 29%; platelets became insensitive to adenosine diphosphate (ADP) and to epinephrine; and plasma levels of low-affinity platelet factor 4 (LA-PF4) progressively rose to 7.4 microgram per milliliter. With PGE1 (1.2 micron), platelet counts remained stable at 92%; platelet reactivity remained normal for 1 hour; and plasma levels of LA-PF4 rose to only 3.3 microgram per milliliter. In rhesus monkeys that underwent cardiopulmonary bypass using a bubble oxygenator without PGE1, platelet counts, expressed as a percent of the prebypass platelet count, declined to 38%; platelets became insensitive to ADP; plasma levels of LA-PF4 progressively rose to 8.4 microgram per milliliter; and the mean postoperative bleeding time was 4.6 minutes. In monkeys that received PGE1, platelet counts declined to only 65%; platelets remained threefold more sensitive to ADP; platelets demonstrated delayed release of LA-PF4 and the mean postoperative bleeding time was 2.7 minutes. This report demonstrates that in a bubble oxygenator system, PGE1 reduces platelet loss, mitigates platelet injury, and shortens postoperative bleeding times following extracorporeal circulation.

Animals↗