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Biomedical subjects

R W Colman

Publications and source records attributed to R W Colman.

At least 307 records · Page 17Linked to original sources
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

Contribution of plasma protease inhibitors to the inactivation of kallikrein in plasma.

Although Cl-inhibitor (Cl-INH) and alpha(2)-macroglobulin (alpha(2)M) have been reported as the major inhibitors of plasma kallikrein in normal plasma, there is little quantitative support for this conclusion. Thus, we studied the inactivation of purified kallikrein in normal plasma, as well as in plasma congenitally deficient in Cl-INH, or artificially depleted of alpha(2)M by chemical modification of the inhibitor with methylamine. Under pseudo-first-order conditions, the inactivation rate constant of kallikrein in normal plasma was 0.60 min(-1). This rate constant was reduced to 0.35, 0.30, and 0.06 min(-1), in plasma deficient respectively in Cl-INH, alpha(2)M, or both inhibitors. Thus Cl-INH (42%) and alpha(2)M (50%) were found to be the major inhibitors of kallikrein in normal plasma. Moreover all the other protease inhibitors present in normal plasma contributed only for 8% to the inactivation of the enzyme. To confirm these kinetic results, (125)I-kallikrein (M(r) 85,000) was completely inactivated by various plasma samples, and the resulting mixtures were analyzed by gel filtration on Sepharose 6B CL for the appearance of (125)I-kallikrein-inhibitor complexes. After inactivation by normal plasma, 52% of the active enzyme were found to form a complex (M(r) 370,000) with Cl-INH, while 48% formed a complex (M(r) 850,000) with alpha(2)M. After inactivation by Cl-INH-deficient plasma, >90% of the active (125)I-kallikrein was associated with alpha(2)M. A similar proportion of the label was associated with Cl-INH in plasma deficient in alpha(2)M. After inactivation by plasma deficient in both Cl-INH and alpha(2)M, (125)I-kallikrein was found to form a complex of M(r) 185,000. This latter complex, which may involve antithrombin III, alpha(1)-protease inhibitor, and/or alpha(1)-plasmin inhibitor, was not detectable in appreciable concentrations in the presence of either Cl-INH or alpha(2)M, even after the addition of heparin (2 U/ml). These observations demonstrate that Cl-INH and alpha(2)M are the only significant inhibitors of kallikrein in normal plasma confirming previous predictions based on experiments in purified systems. Moreover, in the absence of either Cl-INH or alpha(2)M, the inactivation of kallikrein becomes almost entirely dependent on the other major inhibitor.

Angioedema↗
PubMed1982

Inactivation of factor XIa by plasma protease inhibitors: predominant role of alpha 1-protease inhibitor and protective effect of high molecular weight kininogen.

Factor XIa is a plasma protease that, by activating Factor IX, plays an important role in the early phase of the intrinsic pathway of blood coagulation. Four plasma protease inhibitors, alpha(1)-protease inhibitor, antithrombin III, C1-inhibitor, and alpha(2)-plasmin inhibitor, have been reported to inactivate human Factor XIa, but their quantitative contribution to the inactivation of Factor XIa in plasma has not been fully assessed. Using purified systems, we observed that the second-order rate constants for the reaction of Factor XIa with alpha(1)-protease inhibitor, antithrombin III, and CI-inhibitor were 4.08, 10, and 14.6 M(-1) min(-1) x 10(3), respectively. The pseudo-first-order rate constants, at plasma concentration of the inhibitors, were 1.86 x 10(-1), 4.68 x 10(-2), and 2.4 x 10(-2) min(-1), respectively. These kinetic data predict that alpha(1)-protease inhibitor should account for 68%, antithrombin III for 16%, and C1-inhibitor and the equipotent alpha(2)-plasmin inhibitor each for 8% of the total inhibitory activity of plasma against Factor XIa. The rate of inactivation of Factor XIa in various plasma samples specifically deficient in inhibitors was consistent with these predictions. Factor XI, the zymogen form of Factor XIa, circulates in plasma associated with the contact system cofactor, high molecular weight kininogen (HMW kininogen). Kinetic analysis indicated the existence of a reversible bimolecular Factor XIa-HMW kininogen complex with a dissociation constant (K(d)) = 0.17 muM. The light chain derived from HMW kininogen decreased the inactivation rate of Factor XIa by C1-inhibitor with a K(d) of 0.08 muM for a complex of Factor XIa and the light chain derived from HMW kininogen. The protective effect of HMW kininogen was confirmed by the finding that the inactivation rate of Factor XIa in kininogen-deficient plasma was increased over normal plasma. The present study confirms that alpha(1)-protease inhibitor is the major inhibitor of Factor XIa in plasma, and that the formation of a reversible complex between Factor XIa and HMW kininogen decreases the rate of inactivation of the enzyme by its inhibitors.

Antithrombin III↗
PubMed1982

Purified human plasma kallikrein aggregates human blood neutrophils.

Exposure of human blood polymorphonuclear leukocytes (PMN) to purified active plasma kallikrein resulted in PMN aggregation when kallikrein was present at concentrations ranging from 0.4 to 0.6 U/ml (0.18-0.27 microM). Kallikrein-induced PMN aggregation was not mediated through C5-derived peptides, because identical responses were observed whether or not kallikrein had been preincubated with an antibody to C5. Moreover, kallikrein was specific for aggregating PMN, because no aggregation was observed with Factor XII active fragments (23 nM), Factor XIa (0.6 U/ml or 15nM), thrombin (1.6 microM), plasmin (2 microM), porcine pancreatic elastase (2 microM), bovine pancreatic chymotrypsin (2 microM), or bradykinin (1 microM). Bovine pancreatic trypsin (2 microM) aggregated PMN, but to a lesser extent than kallikrein (0.18 microM). Kallikrein was a potent aggregant agent for PMN because similar responses were observed with kallikrein (0.5 U/ml or 0.23 microM) and an optimal dose (0.2 microM) of N-formyl-methionyl-leucyl-phenylalanine. In addition, PMN incubation with kallikrein resulted in stimulation of their oxidative metabolism as assessed by an increased oxygen uptake. Neutropenia and leukostasis observed in diseases associated with activation of the contact phase system may be the result of PMN aggregation by plasma kallikrein.

Cell Aggregation↗
PubMed1982

New and rapid functional assay for C1 inhibitor in human plasma.

C1 inhibitor (C1-INH) and alpha 2-macroglobulin (alpha 2M) account for over 90% of the inactivation of purified plasma kallikrein by normal human plasma. The rate of kallikrein inactivation is also dependent on the presence of high molecular weight kininogen (HMWK), which forms a reversible complex with kallikrein protecting the active site of the enzyme against inhibitors. By selectively inactivating alpha 2M with methylamine, and eliminating the protective effect of HMWK by dilution, the inactivation of kallikrein by plasma became almost exclusively dependent on C1-INH. Functional C1 inhibitor was assessed by measuring the pseudo-first-order rate constant for the inactivation of kallikrein by diluted methylamine-treated plasma in 29 individuals, including 11 controls, 11 oral contraceptive users, 5 patients with classical hereditary angioedema (HAE), and 2 patients with variant HAE. Over a wide range of concentrations, an excellent correlation (r = 0.90) was observed between functional and antigenic C1-INH among controls, oral contraceptive users, and patients with classical HAE. This new functional assay for C1-INH can be performed in less than 3 hr with commercially available reagents. Therefore, this assay will be helpful for the diagnosis and management of conditions associated with the deficiency of C1-INH, such as HAE.

Angioedema↗
PubMed1982

Functional alpha 1 protease inhibitor produced by a human hepatoma cell line.

Alpha 1 protease inhibitor antigen was identified in the culture medium of the human ascites hepatoma cell line SK-HEP-1. Trypsin inhibitory activity and alpha 1 Pl antigen accumulated in serum-free medium concomitantly over a period of several days. Radioactive alpha 1 Pl antigen was detected in conditioned medium from cultures supplemented with 35S-L-methionine, indicating a synthesis and release of the protein. Alpha 1 Pl antigen in conditioned medium appeared to be antigenically identical to that in human plasma, and the newly synthesized (radiolabeled) antigen co-migrated with plasma, alpha 1 Pl after immunoelectrophoresis or SDS-polyacrylamide gel electrophoresis. Moreover, evidence is presented that the synthesized inhibitor exhibits functional activity, since the 35S-labeled alpha 1 Pl in conditioned medium complexes with trypsin. We conclude that SK-HEP-1 cells in culture produce functionally active alpha 1 Pl which may be identical to that in plasma.

Animals↗
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

Factor XI antigen and activity in human platelets.

Washed platelets, contaminated with less than 0.20% plasma factor XI, were examined for the presence of factor XI antigen and activity. These platelets contained a factor-XI-like coagulant activity (0.67 +/- 0.11 U/10(11) platelets) that remained constant after successive washes. By means of indirect immunofluorescence, a monospecific antibody to factor XI showed specific staining of both normal platelets and platelets from patients deficient in plasma factor XI. Radiolabeled Triton extracts of washed platelets and labeled purified factor XI solutions were analyzed for factor XI antigen by Staph A immunoprecipitation analysis using antibody to purified plasma factor XI followed by SDS gel electrophoresis. On unreduced gels, the platelet material ran as a single band having an apparent molecular weight of 220,000 daltons, whereas purified plasma factor XI gave a single band at 160,000 daltons. On reduced gels, the platelet material analyzed as a single band at 52,000 daltons, whereas purified factor XI gave a single band of 80,000 daltons. Analysis of a partially purified factor XI preparation from platelets by immunoelectrophoresis revealed that the platelet preparation displayed a slightly lower cathodal electrophoretic mobility at pH 8.6 than did plasma factor XI and yet appeared to possess complete antigenic identity with plasma factor XI. These results indicate that platelets possess a form of factor XI that exists as a disulfide-linked 52,000-dalton tetramer in contrast to the plasma form that circulates as a 80,000-dalton disulfide-linked dimer.

Antigens↗
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↗
PubMed1982

Effect of heparin on the inactivation rate of human factor XIa by antithrombin-III.

Factor XIa catalyzes an important reaction in the early phase of blood coagulation by converting factor IX to an active enzyme (factor IXa). Although antithrombin-III, an inhibitor of factor XIa, normally accounts for only one-sixth of the plasma inhibitory activity against factor XIa, its effectiveness has been reported to be enhanced by heparin. We have reinvestigated the ability of heparin to potentiate factor XIa inhibition by both purified antithrombin-III and plasma using synthetic tripeptide amide substrates as well as a coagulant assay. No increase in the inactivation rate of factor XIa amidolytic activity by purified antithrombin-III was observed in the presence of therapeutic heparin concentrations (1 U/ml), although inhibition of the amidolytic activity of thrombin by purified antithrombin-III was enhanced at least 20-fold by the same concentration of heparin. Furthermore, despite the ability of heparin (1 U/ml) to increase the inactivation rate of thrombin by plasma, no acceleration of the rate of inhibition of factor XIa by plasma was observed. Similar results were found when the inhibition of factor XIa was monitored with a coagulant assay after first removing the heparin. Only at heparin concentrations of 5 and 10 U/ml, was a 2- and 4-fold increase in the inactivation rate of factor XIa by purified antithrombin III observed. Therefore, in both purified systems as well as plasma, heparin, at concentrations observed in clinical practice, does not accelerate the inactivation rate of human factor XIa by antithrombin-III.

Amides↗
PubMed1981

Affinity labeling of a human platelet membrane protein with 5'-p-fluorosulfonylbenzoyl adenosine. Concomitant inhibition of ADP-induced platelet aggregation and fibrinogen receptor exposure.

Incubation of washed human blood platelets with 5'-p-fluorosulfonylbenzoyl [3H]adenosine (FSBA) covalently labels a single polypeptide of Mr = 100,000. Protection by ADP has suggested that an ADP receptor on the platelet surface membrane was modified. The modified cells, unlike native platelets, failed to aggregate in response to ADP (100 microM) and fibrinogen (1 mg/ml). The extent of binding of 125I-fibrinogen and aggregation was inhibited to a degree related to the incorporation of 5'-p-sulfonylbenzoyl adenosine (SBA) into platelets, indicating FSBA could inhibit the exposure of fibrinogen receptors by ADP necessary for aggregation. Incubation of SBA platelets with alpha-chymotrypsin cleaved the covalently labeled polypeptide and concomitantly reversed the inhibition of aggregation and fibrinogen binding. Platelets proteolytically digested by chymotrypsin prior to exposure to FSBA did not require ADP for aggregation and fibrinogen binding. Moreover, subsequent exposure to FSBA did not inhibit aggregation or fibrinogen binding. The affinity reagent FSBA can displace fibrinogen bound to platelets in the presence of ADP, as well as promote the rapid disaggregation of the platelets. The apparent initial pseudo-first order rate constant of dissociation of fibrinogen was linearly proportional to FSBA concentrations. These studies suggest that a single polypeptide can be altered either by ADP-induced conformational changes or proteolysis by chymotrypsin to reveal latent fibrinogen receptors and promote aggregation of platelets after fibrinogen binding.

Adenosine↗
PubMed1981

Protection of human plasma kallikrein from inactivation by C1 inhibitor and other protease inhibitors. The role of high molecular weight kininogen.

High Mr kininogen increases the activation rate of prekallikrein by activated factor XII on a surface. The resulting serine protease, plasma kallikrein, Mr 88 000, is inhibited in plasma by C1 inhibitor, Mr 105 000. Since prekallikrein circulates in plasma with high Mr kininogen as a complex and a kallikrein-high Mr kininogen complex can be formed in purified systems, we studied whether the inhibition of kallikrein by C1 inhibitor was influenced by high Mr kininogen. With C1 inhibitor in excess, the inactivation of kallikrein followed pseudo-first-order kinetics. The second-order rate constant for the reaction was 1.7 X 10(4) M-1 s-1, and a kallikrein-C1 inhibitor complex, Mr 190 000 was identified on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Kallikrein and C1 inhibitor formed an irreversible complex without measurable prior equilibrium. The rate of this reaction was decreased by 50% in the presence of high Mr kininogen (1 unit/mL or 0.73 muM). Kinetic analysis indicated that this protection was the result of the formation of a reversible complex between kallikrein and high Mr kininogen, which had a dissociation constant of 0.75 muM. However, low Mr kininogen did not protect kallikrein from inactivation by C1 inhibitor. High Mr kininogen also protected kallikrein from inactivation by diisopropyl fluorophosphate. These findings suggest that the kallikrein-high Mr kininogen complex was formed by noncovalent interactions between the light chains of both kallikrein and high Mr kininogen.

Complement C1 Inactivator Proteins↗