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Biomedical subjects

R I Handin

Publications and source records attributed to R I Handin.

At least 73 records · Page 4Linked to original sources
PubMed1981

Relationship between platelet secretion and prothrombin cleavage in native whole blood.

To determine the relationship between platelet secretion and prothrombin conversion in whole blood, the release of platelet factor 4 and the generation of a X(a)-specific cleavage product of prothrombin, fragment 1 + 2, were measured during the coagulation of whole blood. There was a parallel increase in the concentration of the two proteins. Over the first 5 min of incubation, platelet factor 4 concentration increased 6 ng/ml per min, and after 6-7 min, the rate of release increased to 750 ng/ml per min. Over the initial 5-7 min of incubation, fragment 1 + 2 concentration increased 1.5 pmol/ml per min with a subsequent increase of 45 pmol/ml per min. Incubation with 10 muM prostaglandin E(1) or 15 muM prostaglandin I(2) inhibited secretion of platelet factor 4 and delayed the onset of the rapid phase of fragment 1 + 2 generation by 8 min, while stimulation of platelet secretion with 1 mug/ml collagen suspension enhanced production of fragment 1 + 2. The addition of either 10 muM epinephrine or 100 ng/ml collagen suspension to whole blood did not affect either platelet factor 4 release or fragment 1 + 2 generation, although the combination of 3 muM epinephrine and 100 ng/ml collagen suspension enhanced platelet release and prothrombin cleavage. The relationship between platelet factor 4 release and prothrombin cleavage was also studied in Factor VIII-deficient blood. When 0.001 U/ml factor VIII activity was present, <80 ng/ml platelet factor 4 were released, and no fragment 1 + 2 was generated after 30 min of incubation. The addition of 0.008-0.08 U/ml Factor VIII activity progressively increased platelet factor 4 release and prothrombin cleavage. Platelet factor 4 release was normal at 0.08 U/ml Factor VIII activity, whereas prothrombin cleavage was still delayed. Very little thrombin, the amount generated by the cleavage of 3-5 nM fragment 1 + 2, was needed to induce release of platelet factor 4.

Aspirin↗
PubMed1981

Prostacyclin and beta-adrenergic catecholamines inhibit arachidonate release and PGI2 synthesis by vascular endothelium.

We have investigated the mechanism by which cyclic AMP inhibits PGI2 synthesis in cultured bovine aortic endothelial cells. Inhibition of cyclic AMP phosphodiesterase activity by 3-isobutyl-1-methylxanthine (IBMX) blocks calcium ionophore-induced PGI2 production by 62%. The addition of 3 mM dibutyryl cyclic AMP, alone with IBMX, increases the inhibition to 96%. Release o 3H-arachidonate from membrane phospholipids was inhibited 25% by dibutyryl cyclic AMP, 48% by IBMX, and 76% by isoproterenol plus IBMX. Inhibition by isoproterenol was reversed by 10 micro M propranolol. Release of 3H-arachidonate was also reduced 75% by a combination of 10 micro M PGI2 and 3 mM IBMX. We conclude that hormones like isoproterenol and PGI2 may regulate endothelial cell PGI2 biosynthesis by increasing intracellular cyclic AMP, which then inhibits release of endogenous arachidonate from membrane phospholipids.

1-Methyl-3-isobutylxanthine↗
PubMed1981

The interaction of platelets with aortic subendothelium: inhibition of adhesion and secretion by prostaglandin I2.

We have studied the effect of prostaglandin I2 on platelet turnover, attachment to the subendothelium, and secretion following balloon deendothelialization of the rabbit aorta. Survival of 51Cr-labeled platelets in the balloon-injured animals remained normal. Thirty minutes after injury, there were 4.52 X 10(6) platelets/sq cm attached to the aortic surface, which was 87% covered by platelets. Although plasma platelet factor 4, as measured by radioimmunoassay, did not rise above the normal level of 6.8 +/- 2.6 ng/ml (mean +/- SEM) during the first hour after balloon injury, platelet factor 4 antigen was detected within the vessel wall by direct immunofluorescence within 30 min of injury. An infusion of 650-850 ng/kg/min prostaglandin I2 completely inhibited platelet aggregation and reduced surface coverage by 84% and platelet attachment by 63%. Animals given 50-100 ng/kg/min prostaglandin I2, which only partially inhibited platelet aggregation, had 70% of the aortic surface covered by platelets. Platelet factor 4 antigen was also detected within the aortic wall. Platelet attachment was normal in animals that had been given 850 ng/kg/min prostaglandin I2 prior to balloon injury but sacrificed after the infusion was stopped and ex vivo platelet aggregation had returned to normal.

Animals↗
PubMed1980

Vascular permeation of platelet factor 4 after endothelial injury.

Antibody to platelet factor 4 was used to demonstrate permeation of this factor into the blood vessel wall after endothelial injury in rabbits. The presence of platelet factor 4 antigen in the vessel wall after removal of the endothelium was shown by immunofluorescence 10 and 30 minutes after injury but not 240 minutes afterward. This study demonstrates that factors carried by platelets can enter the vessel wall and that the movement of these platelet products into the vasculature is a short-lived, self-limiting process.

Animals↗
PubMed1980

Platelet activation during exercise-induced myocardial ischemia.

Because platelet activation may be important in the worsening of coronary atherosclerosis, we used a radioimmunoassay for platelet factor 4 to study platelet behavior in patients with coronary-artery disease. Forty patients had paired blood samples withdrawn for measurement of the plasma level of platelet factor 4 before and after a standardized exercise-tolerance test. Twenty patients had positive tests, and 19 of those 20 had clinical or angiographic evidence of coronary-artery disease. Eleven of the 20 had a greater than 50 per cent increase in platelet factor 4 after exercise. The remaining nine had positive exercise tests without rises in platelet factor 4. Elevated levels returned to normal within 15 minutes of exercise. Eighteen of 20 patients with negative exercise tests had no rise in platelet factor 4 after exercise. We conclude that a subset of patients with coronary-artery disease and exercise-induced myocardial ischemia had evidence of platelet activation and secretion. (N Engl J Med 302:193-197, 1980).

Adolescent↗
PubMed1980

Inhibition of platelet function by organic nitrate vasodilators.

There is evidence that platelet activation in the coronary circulation may be important in the pathogenesis of myocardial ischemia. Since organic nitrate vasodilators are commonly used in coronary artery disease, we have studied the in vitro effects of these drugs on platelet function. Nitroglycerin, isosorbide dinitrate, and their biotransformation product, inorganic nitrite, inhibited platelet aggregation with collagen, epinephrine, arachidonate, and ionophore, and blocked both primary and secondary aggregation in response to ADP. Nitroglycerin was studied in more detail. Its inhibitory effect was reversible and not dependent on external calcium concentration. It inhibited arachidonic acid oxygenation as measured by the arachidonate-induced oxygen burst and malonaldehyde production. These effects were not due to an increase in intracellular cyclic AMP. This unusual generalized inhibition of platelet function by nitroglycerin possibly contributes to its beneficial effect in myocardial ischemia in part by attenuating platelet reactivity in the coronary circulation.

Adenylyl Cyclases↗
PubMed1980

In vitro studies of thromboresistance: the role of prostacyclin (PGI2) in platelet adhesion to cultured normal and virally transformed human vascular endothelial cells.

Circulating blood platelets normally do not adhere to, or aggregate on, the vascular endothelial lining. We have developed an in vitro model system to study the mechanisms of endothelial resistance to platelet adhesion, and to determine the role of prostacyclin (PGI2) in this process. This system combines scanning electron microscopy and measurement of bound (3H)-adenine-labeled platelets to examine platelet adhesion to primary cultures of human endothelial cells, which generate PGI2-like activity, and to virally transformed endothelial cells, which lack this activity. Under basal conditions primary cultures bound less than one platelet per cell (228 +/- 8 c.p.m. per 10(4) cells, mean +/- standard error of the mean). Inhibition of endothelial PGI2 production by 50 microM aspirin or 2.8 microM indomethacin did not result in a significant change in platelet adherence. Stimulating prostaglandin production with arachidonic acid, or adding exogenous PGI2 did not depress platelet adhesion below the basal levels observed with untreated cultures. In contrast ot primary cultures, transformed endothelium showed markedly increased platelet adherence (3,993 +/- 194 c.p.m. per 10(4), mean +/- standard error of the mean), in the form of single platelets and clusters of two to five nonaggregated platelets. Although exogenous PGI2 was effective in hibiting platelet adherence to these transformed cells, even pharmacologic doses (1 microgram. per ml.) did not depress adhesion to the basal levels associated with normal cells. These results suggest that endothelial properties essential to blood compatibility are altered by viral transformation, and further, that generation of PGI2 by normal endothelium is not the key factor which prevents platelet adherence to the intact vessel wall.

Aspirin↗
PubMed1980

Regulation of platelet arachidonic acid oxygenation by cyclic AMP.

Intracellular cyclic adenosine monophosate (AMP) levels regulate the generation of thromboxane by platelets by inhibiting the hydrolysis of arachidonic acid from membrane phospholipids. However, there is conflicting evidence regarding the role of cyclic AMP in the control of the subsequent oxygenation of arachidonic acid by cyclooxygenase. We studoed the regulation of cyclooxygenase activity by agents that elevate platelet cyclic AMP (dibutyryl cyclic AMP and prostaglandins), measuring arachidonate-induced aggregation, O2 consumption, and malonaldehyde formation. In platelet-rich cyclic AMP. This inhibitory effect of cyclic AMP was absent in gel-filtered platelets suspended in buffer containing 0.5% albumin, and was progressively restored as plasma was added in increasing concentrations. Increasing the albumin concentration in platelet buffer suspensions likewise increased the ability of cyclic AMP to block the arachidonate-induced O2 burst and MDA production. We conclude that (1) the presence of plasma proteins is important in investigating platelet plasma milieu or at least in the presence of physiologic albumin concentrations.

Arachidonic Acids↗
PubMed1979

Solubilization and characterization of a platelet membrane ADP-binding protein.

Previous studies have shown that platelet membranes bind radiolabeled ADP and have nucleoside diphosphokinase activity which transforms added ADP to ATP. In order to further characterize these reactions, the ADP-binding and nucleoside diphosphokinase activity of purified platelet membranes were solubilized by freeze-thaw injury followed by extraction with isotonic buffered saline. Up to 80% of membrane ADP-binding activity was solubilized along with 20% of the total membrane protein, a 4-fold purification. A Millipore filter binding assay was developed to detect the soluble binding protein using [3H]ADP as radioligand. Binding of [3H]ADP was rapid, reversible, saturable, and was destroyed by heat, trypsin digestion, and 1 mM N-ethylmaleimide. By Scatchard analysis, there was a single class of binding sites with a Kd of 3.8 x 10(-7) M. Unlabeled nucleotides competed with [3H]ADP with the following potency series: ATP = ADP greater than AMP greater than adenosine. The solubilized nucleoside diphosphokinase activity could be separated from ADP-binding activity by ultracentrifugation on 5 to 20% sucrose density gradients containing 0.6 M KCl suggesting that the activities reside on separate molecules. Hydrodynamic parameters were calculated for the binding protein by gel filtration and ultracentrifugation. The s20,w was 4.1, Stoke's radius 35 x 10(-8)cm, axial ratio (f/fo) 1.09, and the Mr = 61,000. The studies suggest that this platelet ADP-binding protein may act as the receptor for initiating ADP-induced aggregation and release.

Adenosine Diphosphate↗
PubMed1979

Desensitization of prostaglandin-activated platelet adenylate cyclase.

Prostaglandin D2 (PGD2) is one of several prostaglandins that can inhibit platelet aggregation and activate adenylate cyclase. Platelets were exposed to varying concentrations of PGD2 washed, and the adenylate cyclase response to prostaglandins, epinephrine, and sodium fluoride determined. Incubating platelets with 5 x 10(-5) M PGD2 for 2 hr resulted in a 45% decrease in PGD2 activation of adenylate cyclase and a 25% decrease in stimulation by PGE1. Fluoride activation (7-fold) epinephrine inhibition (30%) and basal enzyme activity were unchanged by exposure of the platelets to PGD2. Desensitization was concentration dependent, with loss of enzyme activity first noted when platelets were incubated with 10(-7) M PGD2. Enzyme sensitivity could be partially restored when desensitized platelets were washed free of PGD2 and incubated in buffer for 2 hr; complete resensitization required incubation for 24 hr in plasma. Regulation of prostaglandin sensitive platelet adenylate cyclase could be of importance in mediating the response of platelets to aggregating agents.

Adenylyl Cyclases↗
PubMed1978

Impairment of hemostasis in patients with severe hemophilia. Failure of diphenhydramine, chlorpromazine, and guaifenesin.

Twenty-three patients with severe hemophilia were given single or multiple doses of diphenhydramine hydrochloride, chlorpromazine hydrochloride, or guaifenesin to test their effects on hemostasis. In contrast with earlier studies with aspirin, bleeding times were not prolonged after ingestion of these three drugs. No patient had hemorrhage because of the drugs, and none recalled excessive bleeding following drug ingestion. These results greatly extend the clinical implications of prior observations carried out in normal subjects demonstrating that many antiplatelet drugs have no effects on in vivo hemostasis. We conclude that these drugs are probably safe for patients with bleeding disorders like hemophilia.

Adolescent↗
PubMed1978

Characterization of the human platelet alpha-adrenergic receptor. Correlation of [3H]dihydroergocryptine binding with aggregation and adenylate cyclase inhibition.

Human platelets aggregate and undergo a release reaction when incubated with catecholamines. Indirect evidence indicates that these events are mediated through alpha-adrenergic receptors. We used [(3)H]dihydroergocryptine, an alpha-adrenergic antagonist, to identify binding sites on platelets that have the characteristics of alpha-adrenergic receptors. Catecholamines compete for the binding sites in a stereo-specific manner with the potency series of (-) epinephrine > (-) norepinephrine > (+/-) phenylephrine > (-) isoproterenol. The dissociation constant (K(d)) of (-) epinephrine is 0.34 muM. Binding is saturable using a platelet particulate fraction but not with intact platelets. There are 0.130 pmol binding sites per milligram protein in fresh platelet membranes. This number represents approximately 100 binding sites per platelet. The K(d) for [(3)H]-dihydroergocryptine was 0.003-0.01 muM. The alpha-adrenergic antagonist phentolamine (K(d) = 0.0069 muM) was much more potent than the beta-adrenergic antagonist (+/-) propranolol (K(d) = 27 muM) in competing for the binding sites. The binding data were correlated with catecholamine-induced platelet aggregation and inhibition of basal and prostaglandin E(1)-stimulated adenylate cyclase. (-) Epinephrine was more potent than (-) norepinephrine in producing aggregation whereas (-) isoproterenol was ineffective. The threshold dose for inducing aggregation by (-) epinephrine was 0.46 muM. Phentolamine and dihydroergocyrptine blocked this response, whereas (+/-) propranolol had no effect. (-) Epinephrine and (-) norepinephrine inhibited basal and prostaglandin E(1)-stimulated adenylate cyclase in a dose-related manner. The concentration of (-) epinephrine inhibiting adenylate cyclase 50% was 0.7 muM. This inhibition was also blocked by phentolamine and dihydroergocryptine but not by (+/-) propranolol. The specificity of binding and the close correlation with alpha-adrenergic receptor-mediated biochemical and physiological responses suggest that the [(3)H]dihydroergocryptine binding site represents, or is closely related to, the human platelet alpha-adrenergic receptor. The ability to assay this receptor directly and to correlate these data with independently measured sequelae of receptor activation should facilitate increased understanding of the physiology and pathophysiology of the human platelet alpha-adrenergic receptor.

Adenylyl Cyclase Inhibitors↗
PubMed1978

Effect of corticosteroid therapy on the phagocytosis of antibody-coated platelets by human leukocytes.

Patients with idiopathic thrombocytopenic purpura (ITP), a disorder in which antibody coated platelets are cleared from the circulation by phagocytic cells, are often treated with glucocorticoids. The effect of corticosteroids on the recognition and ingestion of sensitized platelets by phagocytes can be quantified in these patients and compared to changes in platelet levels. Six patients with ITP were treated with 96 mg daily of methylprednisolone for 5 days. This treatment raised their platelet count and simultaneously decreased the ability of their granulocytes to phagocytize antibody-coated platelets and C3-coated paraffin oil droplets. Corticosteroid treatment did not affect the binding of antibody to platelets or the quantity of antibody in the patients' serum. The ingestion defect was present in isolated, washed leukocytes and persisted for 3-5 days after the corticosteroids were discontinued. Granulocytes and purified monocytes obtained from patients with other medical disorders receiving corticosteroids also ingested paraffin oil droplets and opsonized platelets at a slower rate. These studies provide direct evidence that corticosteroids induce a generalized phagocytic defect and that this may be the mechanism by which corticosteroids raise the platelet count in patients with ITP.

Adrenal Cortex Hormones↗