Biomedical subjects
R W Colman
Publications and source records attributed to R W Colman.
Hyperacute renal allograft rejection in the primate. Therapeutic limitations of antiplatelet agents alone and combined with heparin.
Two antiplatelet drugs, pyridinolcarbamate and Sudoxicam, were tested separately and in combination with heparin for their ability to modify experimental hyperacute renal allograft rejection in primates. Pyridinolcarbamate delayed and amerliorated tissue injury and obstructive thrombosis but only minimally prolonged renal blood flow over that seen in untreated allografts, suggesting that graft failure was primarily due to vasoconstriction. Sudoxicam, an antiinflammatory agent, resulted in higher initial blood flow, but the duration of flow and graft survival were again only minimally prolonged. However, functional changes including C3, Factor II and X consumption were prevented, a net increase in Factor VIII activity was minimized, and fibrinolysis was inhibited. The combined effects of pyridinolcarbamate and heparin resembled those of heparin alone. Combination of Sudoxicam with heparin was more effective than heparin along in preventing intrarenal complement consumption, sequestration of formed elements, activation of coagulation, and inhibition of fibrinolysis. However, the latter combination also failed to prolong venous flow and graft survival over that seen with heparin alone.
Successful short-term modification of hyperacute renal allograft rejection in the primate. Intrarenal effects of phenoxybenzamine and methylprednisolone combined with heparin.
Inhibition of renal vasoconstriction during hyperacute rejection by phenoxybenzamine or methylprednisolone combined with either the antiplatelet agent pyridinolcarbamate or heparin was evaluted in primates. Phenoxybenzamine plus pyridinolcarbamate did not prolong kidney survival. Phenoxybenzamine plus heparin uniformly prolonged low rates of venous flow to 180 minutes and delayed secondary C3 consumption, sequestration of erythrocytes and platelets, coagulation, and fibrinolysis; neutrophil sequestration and vascular injury and obstruction were more marked than with heparin alone. Host pretreatment with methylprednisolone plus heparin also prolonged the low rates of venous flow to 180 minutes, further reduced secondary alterations, and resulted in the least vascular injury. When intact donor kidneys were also pretreated with methylprednisolone, persistently normal rates of venous flow were achieved. Despite marked consumption of Factor XII, the consumption of C3, other coagulation factors, prekallikrein, and sequestration of formed elements was minimal, and the histology appeared compatible with even more prolonged survival.
Effect of heterologous antibody on human platelets.
The effect of heterologous anti-human platelet antibody on human platelet function was examined in the presence and absence of whole plasma as an in vitro model for antibody-induced immune damage to cells. Heterologous IgG anti-human platelet antibody mediated platelet aggregation and released serotonin from both platelets in plasma and from platelets isolated by gel filtration and increased the availability of platelet acid phosphatase in a dose-response fashion. Anti-platelet antibody failed to release beta-glucuronidase (lysosomal enzyme marker) or cause lactic dehydrogenase loss (cytolysis). The effect of the antiplatelet antibody on platelets proceeded in the absence of complement. The active molecule in the anti-platelet antiserum was isolated in the IgG fraction and all three indicators of platelet injury were mediated by purified monomeric IgG. Thrombin was not required for the antibody-mediated effects, as three thrombin inhibitors failed to block the reaction. EDTA was an effective inhibitor, suggesting a cation requirement; however, as little as 38 muM calcium was sufficient for effective platelet aggregation and release. The inability of acetylsalicylic acid to inhibit the effect of the antiplatelet antibody suggests that heterologous antibody (IgG) induced platelet alteration proceeds by a different mechanism than that mediated by ADP and epinephrine and does not involve endogenous platelet prostaglandin synthesis.
Potassium uptake and release by human blood platelets.
Thrombin is known to reduce the K+ content of human platelets, but the subcellular origin of the lost K+ is not known. The effect of aggregating agents on K+ release was studied in platelets labeled in plasma by preincubation with 42KCI. Platelets were separated from plasma by gel filtration through Sepharose 2B equilibrated with K+ -free Tyrode's buffer. Platelet K+ was 116nEq/10(8) platelets, of which 23% was found to be extracellular immediately after gel filtration. K+ influx was 65 nEq/10(8) platelets/hr at pH 7.5 and was more rapid at pH 7.9. About 70% of cell K+ exchanged with plasma in 4 hr with first-order kinetics, while a minor fraction of about 30% exchanged with a slower time course. This slowly exchanging fraction of platelet K+ was thought to arise from heterogeneity in the platelet population. Epinephrine and ADP aggregated gel-filtered platelets and released serotonin, but with loss of only 5%-10% of cell K+ and no beta-glucuronidase. In contrast, thrombin released up to 30% of platelet K+, whether aggregation occurred or was prevented by not stirring the cells. The specific activity of K+ released by all aggregating agents was identical to the specific activity of total platelet K+. Thrombin (0.01-0.2 NIH U/ml) released serotonin and also beta-glucuronidase (an enzyme of the alpha-granule), and there was a linear relation between release of K+ and this enzyme (r = 0.88). No lysis of platelets occurred, since lactic dehydrogenase was not detected. Pretreatment of platelets with aspirin in vitro inhibited thrombin-induced release of serotonin but had no effect on the loss of K+ or beta-glucuronidase. In contrast, the ingestion of aspirin by mouth inhibited the release of serotonin, beta-glucuronidase, and K+ by thrombin. The data suggested that the K+ loss induced by thrombin was primarily derived from release of alpha-granules and that these organelles contained about 20% of the total platelet K+ in a freely exchangeable and nonsequestered state.
Halofenate: a potent inhibitor of normal and hypersensitive platelets.
Platelet hypersensitivity has been documented in diabetes and angina pectoris and can be partially reversed in hyperbetalipoproteinemia by clofibrate. We therefore examined the effects of incubating another lipid-lowering agent, halofenate, with both normal platelets and platelets made hypersensitive in vitro by incorporation of 55 per cent excess cholesterol into their membranes. At therapeutic concentrations, halofenate caused a time- and dose-dependent inhibition of the aggregation of normal platelets by epinephrine. After 30 minutes' incubation at 37 degrees C., halofenate significantly inhibited the extent of aggregation by 88 per cent (p less than 0.01), whereas clofibrate inhibited aggregation by 44 per cent (p less than 0.01). Halofenate was a more potent inhibitor of platelets than clofibrate (p less than 0.01). The mean threshold concentration of epinephrine necessary for aggregation of normal platelets (4.2 muM) was not significatnly increased with clofibrate (10 muM) but was markedly elevated with halofenate (245 muM; p less than 0.001). Significant but less dramatic increases in threshold concentration of ADP and collagen were found with halofenate but no clofibrate. Cholesterol-rich platelets were 114-fold more sensitive to epinephrine and twofold more sensitive to ADP than normal platelets but after incubation with halofenate became even less sensitive than normal. Clofibrate inhibited the extent of aggregation of hypersensitive platelets but did not alter the threshold concentration of epinephrine necessary for aggregation. Thus, halofenate is more potent than clofibrate in reducing the sensitivity of normal platelets to aggregating agents in vitro and can completely reverse experimentally produced platelet hypersensitivity. These data suggest that halofenate might be useful in reversing increased platelet sensitivity in cardiovascular diseases.
Kinin activation in the blood of patients with sepsis.
To determine whether or not kinin activation in the blood during severe infection with gram-negative bacteria may be related to hemodynamic abnormalities encountered, blood prekallikrein, kallikrein inhibitor and kinin values in 2l surgical patients with sepsis were compared with those in normotensive and hypotensive states. Because of reduced prekallikrein synthesis in patients with hepatic insufficiency, the normotensive and hypotensive groups were each subdivided according to the presence or absence of liver dysfunction, as indicated by elevated blood bilirubin, serum glutamic-oxalacetic transaminase or alkaline phosphatase levels. The mortality was zero in group 1, normotensive normal liver function; 80 per cent in group 2, hypotensive-normal liver function; 20 per cent in group 3, normotensive liver dysfunction, and 67 per cent in group 4, hypotensive liver dysfunction. Ultimately, the majority of deaths were due to respiratory failure. Although the blood prekallikrein level, was below normal in all groups and was significantly less in all patients with liver dysfunction, it was reduced proportionately in hypotensive patients to less than 30 per cent of the values noted in the two normotensive groups. This finding suggests prekallikrein consumption in the hypotensive groups to be the result of the process of activating kallikrein and bradykinin. This concept is supported by finding elevated kinin values, above 3 nanograms per milliliter of plasma, in only 28 per cent of those in group 1 and 12 per cent of those in group 3, while in the hypotensive patients, groups 2 and 4, the kinin level was elevated in 60 and 66 per cent, respectively.
Postoperative disseminated intravascular coagulation and fibrinolysis.
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Role of galactose in bovine factor V.
Using galactose oxidase as well as beta-galactosidase to produce modifications of the galactose units, the functional significance of these carbohydrate residues on the coagulant activity of bovine Factor V glycoprotein was evaluated. Incubation of native Factor V with galactose oxidase or hydrolysis of asialo-Factor V with beta-galactosidase results in a loss of Factor V activity. The inactivation of Factor V by oxidation of galactose moieties is partially reversible upon reduction of the newly formed aldehyde groups with sodium borohydride. The extent of reversal depends upon the degree of inactivation achieved. Thus, Factor V which retained 30% of the original activity following galactose oxidation returns to 75% of the original coagulant activity upon borohydride reduction; but, after destruction of 85% of the original activity treatment with borohydride returns to about 30%. In the initial stages of the inactivation of Factor V by treatment with galactose oxidase, the loss of Factor V coagulant activity is directly proportional to the moles of galactose oxidized. However, as the reaction progresses, the rate of galactose oxidation exceeds the rate of loss of Factor V activity. Moreover, galactose oxidation continues even after complete inactivation of Factor V. These results suggest that the galactose residues most susceptible to attack by galactose oxidase are those necessary for the activity of this coagulant protein. Only 15 galactose residues/mol of Factor V are susceptible to galactose oxidase prior to removal of sialic acid. In contrast, 37 galactose residues/mol of Factor V are found after acid hydrolysis. These results suggest that Factor V glycoprotein contains more than one type of sialyl-galactose linkages, the C2,3 or C2,4 linkages susceptible to oxidation in the native protein and the C2,6 linkage which is resistant. Native Factor V binds with diarachidonyl lecithin forming an active complex of lower buoyant density, while the Factor V oxidized with galactose oxidase does not. The Factor V-phospholipid complex is protected from inactivation by galactose oxidase. Moreover, lipid binding diminishes the extent of oxidation of galactose residues. Certain galactose groups are essential for coagulant activity probably because they are required for binding to phospholipid, a prerequisite to Factor V action.
Molecular changes associated with proteolysis of bovine factor V by thrombin.
Native factor V contains two major polypeptide chains, h and 1. The molecular weights determined by gel electrophoresis in the presence of sodium dodecylsulfate and dithiothreitol (125 000 and 73 000) are in reasonable agreement with those obtained by gel filtration in 5 M guanidine-HC1 (125000 and 64000). Exposure of factor V to thrombin results in cleavage of the heavier chain to an altered form with a molecular weight of 87000. The other fragment of this proteolytic reaction appears to be a carbohydrate-rich piece, which migrates abnormally slowly on gel electrophoresis conducted under denaturing and reducing conditions. Both proteolytic cleavage products remain associated with the light chain during the purification of factor V. The 87000-Mr fragment is present in samples of factor V which are isolated by immunoprecipitation of blood obtained from a single animal by venous catheter. This finding suggests that some proteolysis may occur in vivo. In contrast, the molecular weight of the light chain is unaltered after thrombin proteolysis of either purified factor V or thrombin-treated plasma.
Immunochemical studies on factor V.
Native bovine factor V exhibits a molecular weight of 300000 as determined by gel filtration of untreated plasma. Highly purified factor V exhibits multiple molecular weight forms which range from small active fragments to aggregates of several million which are generated during the purification on cellulose phosphate. Isoelectric focusing on a single high-molecular-weight species produced a single protein and activity peak at pH 4.65. Factor V activity is associated with each protein band observed following polyacrylamide gel electrophoresis. Antisera to factor V prepared in rabbits produces a time-dependent and concentration-dependent inhibition of factor V activity in plasma and purified factor V. The multiple molecular weight forms of factor V appear equivalent upon immunodiffusion and on immunoelectrophoresis migrate as an alpha globulin between albumin and fibrinogen. Immunoprecipitation arcs are equivalent in plasma and serum. Factor V consists of two major types of subunits, a light chain (73000), aggregates of which form the high-molecular-weight species, and a heavy chain (125 000). Using preparations containing one or both chains isolated by disc gel electrophoresis, antiserum was shown to contain two families of antibodies, one against each subunit. Cross reactivity with both light and heavy chain antigens is observed in sheep and goat but not monkey or human plasma. The antisera also neutralized goat and sheep factor V activity.
Determinants of the formation and activity of factor V-phospholipid complexes. II. Molecular properties of the complexes.
The stability, buoyancy, and intrinsic activity for factor V-phospholipid complexes were investigated. The decay of factor V activity in the absence of phospholipid followed first order kinetics; however, in the presence of several phospholipids a biphasic decay curve was observed. The addition of phosphatidylethanolamine to factor V produced only a small loss of activity in the first 2 minutes but decreased the subsequent rate of inactivation fourfold. A PE-factor V complex with a low bouyant density was separated from uncomplexed factor V by sucrose density ultracentrifugation. The association constant for this complex was 5 X 10(6) M-1 with approximately 2 moles of factor V bound per mole of lipid micelle. The isolated complex was capable of increasing prothrombin conversion 10-fold without additional phospholipid. A still lighter complex increased the rate of prothrombin conversion 18-fold. Phosphatidyl serine produced a concentration-dependent loss of up to 95% of the factor V activity in the first 2 minutes. After ultracentrifugation on a sucrose density gradient, a PS-factor V complex of increased density was detected. This complex failed to accelerate prothrombin conversion in the intrinsic two-stage assay. Except at very high concentrations, phosphatidylcholine did not alter the kinetics of inactivation of factor V. A factor V-phosphatidylcholine complex could not be detected after ultracentrifugation. When added to factor V, cardiolipin (200 mug/ml), produced a rapid 50% decline in activity with a subsequent three-fold increase in the rate of inactivation. No activity was recovered after ultracentrifugation of factor V in the presence of cardiolipin. Saturated phosphatidylethanolamine produced a concentration dependent initial loss of activity, but only a minimal increase in the subsequent rate of inactivation. At 200 mug/ml almost no light complex was detected after ultracentrifugation, but at 800 mug/ml a light complex was observed. This behavior corresponds to the ability of saturated phosphatidylethanolamine to accelerate prothrombin conversion only at very high concentrations. Thus, phospholipids combine with factor V to form complexes which differ in their ability to accelerate prothrombin conversion. The most active species are stable lipoprotein complexes of lower buoyant density than factor V.
Determinants of the formation and activity of factor V-phospholipid complexes. I. Influence of phospholipid structure.
Previous studies showed that factor V consists of multiple oligomeric forms including a minor component (form L) which contained lipid and was eluted in the excluded volume of a Sepharose 4B column. In the present and following study the hypothesis that factor V is a lipid requiring protein rather than a lipoprotein has been tested. Modification of a previous purification procedure resulted in the separation of factor V from lipoprotein. The lipid extracted from this lipoprotein was qualitatively similar to that previously isolated from form L. Removal of this lipoprotein by sucrose density gradient ultracentrifugation resulted in the isolation of factor V of high specific activity which contained no detectable phospholipid and less than 0.2% cholesterol. No component was excluded from Sepharose 4B after gel filtration of this purified preparation. It is concluded that factor V is not a lipoprotein, but rather a protein which requires lipid for its coagulant properties. To define this requirement further the ability of factor V-phospholipid complexes to accelerate the conversion of prothrombin to thrombin by factor Xa in the presence of calcium was tested. The rate of thrombin formation was highly dependent on the phospholipid employed with each active phospholipid exhibiting a characteristic optimal concentration. At 250 muM, the order of activity was phosphatidyl ethanolamine greater than phosphatidyl inositol greater than cardiolipin greater than phosphatidyl choline. Phosphatidyl serine was inert at all concentrations employed. No single fatty acid was consistently present in active lipid preparations, nor was a specific fatty acid absent from those which had no clotting activity. In comparing different lipid classes, a direct relationship between the overall degree of unsaturation and activity was not observed. However, bovine phosphatidyl inositol, which is highly unsaturated, is active, while plant phosphatidyl inositol, which contains only trace amounts of unsaturated fatty acids, were inert. At lipid concentrations below 300 muM, saturated phosphatidyl ethanolamine produced by hydrogenation of bovine phosphatidyl ethanolamine was less active than an equal concentration of the native lipid. In mixtures containing both lipids, artificially saturated phosphatidyl ethanolamine potentiated the activity of the native compound.
Platelet hypersensitivity and intravascular coagulation in paroxysmal nocturnal hemoglobinuria.
The patient described had paroxysmal nocturnal hemoglobinuria associated with recurrent arterial as well as venous thrombosis. Study of platelet function revealed hypersensitivity to epinephrine, adenosine 5'phosphate (ADP) and collagen as judged by their ability to aggregate platelets as well as to release 14C serotonin. The release of total nucleotides was also markedly increased over normal with all aggregating agents. The abnormality was localized to the platelet since aggregation occurred when the patient's platelets were resuspended in normal plasma but not when normal platelets were incubated in the patient's plasma. Presumptive evidence for ongoing intravascular coagulation was an increase in fibrinogen derivatives of heavier molecular weight than the native protein presumably a result of thrombin action. However, factor XII was not activated and fibrinolysis was not increased. Complement component levels and antithrombin concentrations were also normal. The findings in this case suggest that hypersensitive platelets may contribute to the intravascular coagulation that is manifested by the increased incidence of thrombosis in patients with paroxysmal nocturnal hemoglobinuria.
Roles of sialic acid in the function of bovine factor V.
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The effect of chemical or enzymatic modifications upon the ability of collagen to form multimers and to initiate platelet aggregation.
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Statistical comparison of the fibrometer and the Electra 600 for prothrombin time determination.
Determinations of prothrombin time with the semiautomatic fibrometer and with a more automated machine (Electra 600) were compared in regard to reproducibility, accuracy, and speed. Prothrombin times determined for replicate samples with the two machines showed a correlation coefficient of 0.98, but the Electra 600 sensed the clot 0.5 seconds before the fibrometer. The overall coefficients of variation for multiple tests in the normal therapeutic and elevated ranges of prothrombin time were 3.5% for the fibrometer and 2.0% for the Electra 600. The average technician working time was 60% shorter with the fully automated machine than with fibrometer. It is concluded that the automated machine was more accurate and more rapid in determining prothrombin time.
Platelet hypersensitivity induced by cholesterol incorporation.
Platelets from individuals with familial hypercholesterolemia show increased sensitivity to the aggregating atents, epinephrine and ADP. Since the mechanism of this abnormal sensitivity is unknown, we examined, in vitro, the influence of the plasma lipid environment on the function of platelets. The composition of plasma lipids was altered by the addition of sonicated cholesterol-dipalmitoyl lecithin liposomes which were "cholesterol normal" (cholesterol-phospholipid mole ratio [C/P] equals 1.0, "cholesterol rich" (C/P eauals 2.2), or "cholesterol poor" (C/P equals 0). Cholesterol-normal liposomes had no influence on platelet lipids or platelet function. In contrast, after incubation for 5 h at 37 degrees C with cholesterol-rich liposomes, normal platelets acquired 39.2% excess cholesterol with no change in phospholipids or protein. The percent increase in platelet membrane cholesterol was three-fold that of the granule fraction. The acquisition of cholesterol by platelets was associated with a 35-fold increase in sensitivity to epinephrine-induced aggregation (P less than 0.001) and 15-fold increase to ADP aggregation (P less than 0.001), as determined both by aggregometry and by [13C]serotonin release. Response to thrombin or collagen was unchanged. Platelets incubated with cholesterol-poor liposomes underwent a selective loss of 21.4% cholesterol and this was associated with an 18-fold reduction in their sensitivity to epinephrine. These studies demonstrate that the cholesterol content of platelets is dependent on the lipid composition of the milier. Cholesterol acquired by platelets may exert its effect on platelet function by a modification of the platelet membrane.