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Biomedical subjects

R W Colman

Publications and source records attributed to R W Colman.

At least 289 records · Page 16Linked to original sources
PubMed1984

Amidolytic assay of human factor XI in plasma: comparison with a coagulant assay and a new rapid radioimmunoassay.

The traditional coagulant assay for plasma factor XI suffers from a relatively high coefficient of variation, the need for rare congenitally deficient plasma, and a poor correlation between precision and sensitivity. We have developed a simple functional amidolytic assay for factor XI in plasma using the chromogenic substrate PyrGlu-Pro-Arg-p-nitroanilide (S-2366). After inactivation of alpha 1-antitrypsin, CI inhibitor, and other plasma protease inhibitors with CHCI3, plasma was incubated with kaolin, in the absence of added calcium, which limited the enzymes formed to those dependent on contact activation. Soybean trypsin inhibitor was used to minimize the action of kallikrein on the substrate. Once the reaction was complete, corn trypsin inhibitor was used to inactive factor XIIa, the enzyme generated by exposure of plasma to negatively charged surfaces, which had activated the factor XI. The assay is highly specific for factor XI, since plasma totally deficient in that zymogen yielded only 1%-3% of the enzymatic activity in normal plasma under identical conditions. The requirements for complete conversion of factor XI to XIa in plasma within 60 min were, respectively, factor XII, 0.6 U/ml, and high molecular weight kininogen, 0.2 U/ml. Prekallikrein was not an absolute requirement for complete activation but did accelerate the reaction. The intraassay coefficient of variation was 3.4%, and the mean of 35 normal plasmas was 1.00 U +/- 0.24 SD. In addition, a new rapid radioimmunoassay was devised using staphylococcal protein A as the precipitating agent for a complex of factor XI antigen with monospecific rabbit antibody. The mean was 1.01 U +/- 0.30 SD. The correlation coefficients for amidolytic versus coagulant and amidolytic versus radioimmunoassay were r = 0.95 for the former and 0.96 for the latter. Thus, a simple, accurate amidolytic assay and a radioimmunoassay have been devised for measuring factor XI in plasma that correlate well with the coagulant activity of factor XI, as determined in our laboratory.

Animals↗
PubMed1983

Inhibition of ADP-induced platelet aggregation by reduced factor Xa.

Treatment of blood coagulation factor Xa with insolubilized hexyl-agarose derivative of prostaglandin E1 (PGE1) results in the generation of two sulfhydryl groups in the protein molecule. The reduced factor Xa was found to be a potent inhibitor of platelet aggregation and thromboxane A2 synthesis induced by ADP. In contrast to the inhibition of thromboxane formation, the reduced factor Xa had no effect on the formation of PGE2 indicating that thromboxane synthetase might be selectively inhibited by the reduced factor Xa. Incubation with oxidized glutathione reversed the inhibitory activity of factor Xa previously exposed to the insolubilized hormone. Soluble PGE1 also reduces factor Xa, but more slowly than the insolubilized PGE1. PGE1 also exhibits reducing ability as tested with redox dyes. Reduction of factor Xa by dithiothreitol also transformed the coagulation factor into an inhibitor of platelet aggregation and thromboxane A2 formation. These experiments indicate that reduction of factor Xa leads to a reversible alteration of the molecule which inhibits platelet aggregation induced by ADP. This effect of reduced factor Xa is probably mediated through the inhibition of thromboxane A2 synthesis.

Adenosine Diphosphate↗
PubMed1983

Cyclic AMP induces synthesis of prostaglandin E1 in platelets.

Although platelets are known to synthesize small amounts of prostaglandin E1 the control of the formation of this prostanoid has not been investigated. Incubation of human platelet-rich plasma with various compounds which are known to increase cyclic AMP concentration in platelets and inhibit platelet aggregation also increased intracellular prostaglandin E1 synthesis. The prostaglandin E1 was isolated by high pressure liquid chromatography and definitively identified by negative and positive ionization mass spectroscopy. The amounts of prostaglandin E1 formed were proportional to the concentration of cyclic AMP in platelets. Prostacyclin (10 nM) which is the most potent stimulator of cyclic AMP formation increased intracellular cyclic AMP by 4.6 fold and prostaglandin E1 level by 3 fold over the basal levels. Addition of theophylline, a cyclic AMP phosphodiesterase inhibitor, together with prostacyclin increased cyclic AMP concentration 8.7-fold and prostaglandin E1 level 12-fold compared to basal concentrations. Dibutyryl cyclic AMP (2 mM) and 8-bromo cyclic AMP (0.1 mM) increased prostaglandin E1 levels by 3 fold and 2 fold over the basal level, respectively. Prostaglandin D2 (3 microM) when added to platelet-rich plasma increased the cyclic nucleotide levels by 2 fold concomitant with 2 fold increase in prostaglandin E1 concentration. In contrast prostaglandin E2 or prostaglandin F2 alpha which had no effect on cyclic AMP level did not affect the prostaglandin E1 synthesis. Addition of 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, to platelet-rich plasma inhibited both the increase of intracellular prostaglandin E1 and cyclic AMP levels induced by prostacyclin.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1983

Cyclic AMP independent inhibition of platelet aggregation by prostaglandin E1 is mediated through factor Xa.

Normal plasma exposed to an insolubilized omega-NH2-hexyl-agarose derivative of PGE1 inhibits ADP-induced platelet aggregation. To define the plasma macromolecule responsible, we tested plasma congenitally deficient in coagulation factors for this property. Only plasma deficient in factor X failed to inhibit platelet aggregation under these conditions. The inhibition could be restored by reconstituting the defective plasma with bovine or human factor X. Purified factor X or Xa exposed to insolubilized PGE1 also inhibited ADP-induced platelet aggregation. Factor Xa could reverse suggesting that factor Xa was the responsible component. This conclusion was further supported by the ability of heparin to reverse the inhibitory effect of altered factor Xa when the anticoagulant was added simultaneously to platelets. The results were not due to the release of PGE1 from the insoluble derivative. Factor Xa may have a heretofore unrecognized effect on modulating platelet functions.

Alprostadil↗
PubMed1983

Prekallikrein activation and high-molecular-weight kininogen consumption in hereditary angioedema.

Patients with hereditary angioedema lack C-1 inhibitor, a plasma alpha 2-glycoprotein that inhibits both the proteolytic action of C1, the activated first component of the complement system, and the activity of components of the contact phase of coagulation: kallikrein, factor XIa, and factor XIIa. Such patients have been shown to have low levels of C4 and C2, the natural substrates for C-1, but the levels were not correlated with the presence of symptoms. We studied three patients with angioedema for evidence of activation of the contact system and found that during a symptomatic period they had decreased levels of prekallikrein, a substrate for the activated forms of factor XII, and reductions in high-molecular-weight kininogen, a substrate for plasma kallikrein. These observations suggest that zymogens of the contact system are activated during attacks of hereditary angioedema and that some of the clinical manifestations may be mediated through products of this pathway, such as kinins.

Adolescent↗
PubMed1983

The role of platelet factor V in prothrombin conversion.

The contribution of platelet factor V to prothrombin conversion was studied in a purified two-stage system designed to measure the ability of factor V to accelerate prothrombin conversion. When unstimulated gel-filtered platelets (GFP) were substituted for both factor V and phospholipid, thrombin evolution was linear following a long lag time. Gel filtration resulted in considerable phospholipid availability with minimal factor V release. Incubating platelets with collagen in increasing concentrations resulted in marked shortening of the lag time, an increase in the initial rate of thrombin formation, and release of platelet factor V. The inhibition of thrombin formation by preincubation of the platelets with metabolic inhibitors is consistent with previous observations that factor V is released from alpha-granules by collagen in a process requiring metabolic energy. Released platelet factor V added to metabolically inhibited platelets reproduces the acceleration of prothrombin conversion demonstrated in GFP incubated with collagen. Furthermore no acceleration of the clotting time at collagen concentrations used in this study was demonstrated in an assay designed to measure available platelet phospholipid in the presence of excess factor V. The rate of increased thrombin generation produced by collagen stimulation is primarily due to released platelet factor V in the system employed.

Animals↗
PubMed1983

Studies on the renin-angiotensin system in a kininogen-deficient individual.

The physiological responses of the renin-angiotensin system were studied in an individual with kininogen deficiency (patient 1) with absent plasma bradykinin and markedly impaired pre-kallikrein conversion into kallikrein. After sodium depletion, patient 1 had a low plasma renin activity (1.4 pmol of ANG I h-1 ml-1) and a low angiotensin II concentration (36 pg/ml) compared with values in 11 normal individuals (4.0 +/- 0.94 pmol of ANG I h-1 ml-1) and 63 +/- 6 pg/ml respectively). Unlike normal individuals, in the kininogen-deficient subject there was no significant fall of renin activity or angiotensin II after dietary sodium repletion. Intravenous sodium repletion also failed to further decrease plasma renin activity or angiotensin II. The usual two- to three-fold rise in plasma renin activity and angiotensin II observed in normal subjects on assumption of the upright posture after ingestion of 200 mg of sodium/day failed to occur in the kininogen-deficient individual. These data in vivo are in agreement with observations in vitro that once plasma kallikrein forms it may be important in converting prorenin into renin. In the absence of kininogen, activation of prekallikrein to kallikrein is grossly defective, which may in part account for the diminished response of the renin-angiotensin system to changes in sodium balance and posture.

Adult↗
PubMed1983

Inhibition of thromboxane A2 synthesis in human platelets by coagulation factor Xa.

Factor Xa binds to platelets provided that factor Va is present on the platelet surface, an interaction that results in a striking acceleration of the conversion of prothrombin to thrombin. Thrombin then initiates fibrin formation, induces platelet aggregation, and stimulates the intraplatelet synthesis of thromboxane A2 (TXA2). Addition of thrombin (2.4-14.4 nM) to platelet-rich plasma increased the basal level of TXA2, measured as thromboxane B2, from less than 0.5 pmol per 10(8) platelets to (mean +/- SEM) 100 +/- 22 and 250 +/- 10 pmol per 10(8) platelets, respectively. Treatment of platelet-rich plasma with increasing concentrations of factor Xa (1-12 nM) prior to the addition of thrombin progressively inhibited the production of TXA2. Thrombin (9.6 nM), which produced 93% of the maximal formation of TXA2, was inhibited 70% by factor Xa (10 nM). To identify which of these steps in thromboxane synthesis was inhibited by factor Xa, platelets labeled with [14C]arachidonic acid were exposed to thrombin and products of prostaglandin synthesis were separated by thin-layer chromatography. In contrast to the inhibition of TXA2 synthesis, prostaglandin E2 and prostaglandin F2 alpha synthesis were not inhibited suggesting that neither phospholipase(s) nor cycloxygenase was involved. The inhibition of TXA2 formation by factor Xa could be reversed by increasing the molar ratio of thrombin to factor Xa to 5.5. Incubation of platelets with an IgG fraction of a human monoclonal antifactor V antibody, previously shown to inhibit factor Xa binding, was found to block factor Xa inhibition of TXA2 synthesis. The inhibition of TXA2 synthesis requires the presence of the active site serine of factor Xa and is not specific for TXA2 formation induced by thrombin because it is also demonstrable when the agonist is ADP. Further, factor Xa does not require additional plasma components for its action because its inhibitory effects are detected in gel-filtered platelets. The effect of factor Xa was evident at physiological (1.3 mM) calcium concentrations. These results indicate that factor Xa binding to platelets through factor Va not only stimulates thrombin formation but also has a countervailing effect by inhibiting TXA2 formation.

Adenosine Diphosphate↗
PubMed1983

Effect of antiplatelet antibody on platelet shape change, volume, and morphology.

We extended our studies of the effect of antibody on human platelets. Monomeric immunoglobulin G (IgG) (rabbit) antiplatelet antibody was interacted with human platelets in the absence of complement and the early morphological changes monitored. Platelet shape change, assessed by a rheooptical approach, was noted within 20 s and was proportional to antibody concentration. Antiplatelet antibody-induced shape change was also accompanied by a change in apparent platelet volume, as measured in the resistive particle counter. Scanning and transmission electron microscopy confirmed a disk-to-sphere transformation associated with the formation of bulbous, short surface projections or pseudopodia. In contrast, ADP-induced disk-to-sphere transformation was associated with the development of long, thin filopodia emanating from the platelet. These specific effects of antiplatelet antibody may result in altered platelet function.

Adenosine Diphosphate↗
PubMed1983

High-molecular weight kininogen. A secreted platelet protein.

Human platelets were studied immunochemically to determine if they contain high-molecular weight kininogen. On crossed immunoelectrophoresis with total kininogen antisera (antisera that recognizes both high- and low-molecular weight kininogen) extracts of platelets contained total kininogen antigen. Platelet total kininogen antigen showed complete antigenic identity with plasma total kininogen and displayed the same electrophoretic migration as plasma total kininogen. Using antisera monospecific to high molecular weight kininogen, a competitive enzyme-linked immunosorbent assay (CELISA) was developed to directly measure platelet high-molecular weight kininogen. By CELISA, 27-101 ng of high molecular weight kininogen antigen per 10(8) platelets was quantitated in detergent-soluble lysates of washed human platelets from nine normal donors with a mean level of 60 ng +/- 24/10(8) platelets. Plasma high-molecular weight kininogen, either in the platelet suspending medium or on the surface of the platelets, could only account for 5% of antigen measured in the solubilized platelets. On the CELISA, platelet high-molecular weight kininogen was immunochemically identical to plasma and purified high-molecular weight kininogen. Platelet high-molecular weight kininogen was secreted from platelets after exposure to ionophore A23187 (3-15 microM), collagen (5-150 micrograms/ml), and thrombin (1.6 U/ml). Secreted platelet high-molecular weight kininogen did not become a part of the platelet Triton-insoluble cytoskeleton. On cross immunoelectrophoresis secreted platelet total kininogen antigen had a similar electrophoretic migration to plasma total kininogen. Thus, human platelets contain high-molecular weight kininogen that can be secreted from platelets and that may participate in plasma coagulation reactions.

Adult↗
PubMed1983

Heterogeneity of human factor V deficiency. Evidence for the existence of antigen-positive variants.

Functional human Factor V has been purified using a rapid immunoaffinity method. Following barium citrate adsorption of plasma, Factor V was precipitated with polyethylene glycol at a concentration between 5 and 14%. The resulting preparation was applied to a column containing an immobilized immunoadsorbent consisting of an IgG fraction containing a naturally occurring human monoclonal (IgG(4)lambda) antibody with inhibitory activity against human Factor V. The solid phase immunoglobulin quantitatively bound Factor V from human plasma. The bound Factor V was effectively eluted with a Tris buffer pH 7.2 containing 1.2 M NaCl and 1 M alpha-methyl-D-mannoside. The isolated native Factor V with high specific activity (92 U/mg) showed a single band (M(r), 350,000) on both reduced and nonreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Factor V was purified 5,100-fold over plasma with an overall yield of 77%. The purified Factor V when subjected to thrombin activation exhibited an 18-fold increase in coagulant activity. The isolated Factor V neutralized the inhibitory activities of the monoclonal antibody that was used to purify it, as well as the rabbit antibodies produced by immunizing the animals with the purified Factor V. Immunoelectrophoresis of purified Factor V against the polyclonal rabbit antiserum resulted in a single precipitin arc of identical mobility to the Factor V in normal human plasma. Analysis by double immunodiffusion showed a line of identity between plasma and purified Factor V and crossed immunoelectrophoresis showed a single species in normal plasma.A competitive enzyme-linked immunosorbent assay using the rabbit antibody against Factor V was applied to quantify Factor V antigen level in human plasma. Reconstitution of congenitally deficient or immunodepleted plasma with normal plasma or purified Factor V gave parallel dose-response curves. In 14 normal plasma the coagulant activity was 0.98+/-0.02 U/ml (mean+/-SEM) and antigen concentration was 11.1+/-0.4 mug/ml. A pool of 14 patients with congenital Factor V deficiency were studied. 10 patients had Factor V antigen ranging from 1.0 to 2.4 mug/ml with corresponding coagulant activities (0-0.17 U/ml) indicating a low concentration of normal Factor V, presumably due to decreased synthesis or increased degradation. When these patient plasmas and the normal plasmas were analyzed together an excellent correlation (r = 0.97, P < 0.01) was obtained. However, four patients with coagulant activity (0-0.08 U/ml) had Factor V antigen concentrations ranging from 4.4 to 6.1 mug/ml, indicating the presence of a reduced concentration of abnormal Factor V protein. The presence of patients with antigen similar in concentration to coagulant activity and antigen in excess of Factor V activity indicates the heterogeneity of congenital Factor V deficiency.

Animals↗
PubMed1983

Human plasma kallikrein releases neutrophil elastase during blood coagulation.

Elastase is released from human neutrophils during the early events of blood coagulation. Human plasma kallikrein has been shown to stimulate neutrophil chemotaxis, aggregation, and oxygen consumption. Therefore, the ability of kallikrein to release neutrophil elastase was investigated. Neutrophils were isolated by dextran sedimentation, and elastase release was measured by both an enzyme-linked immunosorbent assay, and an enzymatic assay using t-butoxy-carbonyl-Ala-Ala-Pro-Val-amino methyl coumarin as the substrate. Kallikrein, 0.1-1.0 U/ml, (0.045-0.45 microM), was incubated with neutrophils that were preincubated with cytochalasin B (5 micrograms/ml). The release of elastase was found to be proportional to the kallikrein concentration. Kallikrein released a maximum of 34% of the total elastase content, as measured by solubilizing the neutrophils in the nonionic detergent Triton X-100. A series of experiments was carried out to determine if kallikrein was a major enzyme involved in neutrophil elastase release during blood coagulation. When 10 million neutrophils were incubated in 1 ml of normal plasma in the presence of 30 mM CaCl2 for 90 min, 2.75 micrograms of elastase was released. In contrast, neutrophils incubated in prekallikrein-deficient or Factor XII-deficient plasma released less than half of the elastase, as compared with normal plasma. The addition of purified prekallikrein to prekallikrein-deficient plasma restored neutrophil elastase release to normal levels. Moreover, release of elastase was enhanced in plasma deficient in C1-inhibitor, the major plasma inhibitor of kallikrein. This release was not dependent upon further steps in the coagulation pathway, or on C5a, since levels of elastase, released in Factor XI- or C5-deficient plasma, were similar to that in normal plasma, and an antibody to C5 failed to inhibit elastase release. These data suggest that kallikrein may be a major enzyme responsible for the release of elastase during blood coagulation.

Blood Coagulation↗
PubMed1982

The effect of a medicinal Chinese herb on platelet function.

We investigated the effect of the Chinese herb Injectio Salvia Miltiorrhizae (ISM) on human platelet function in vitro. ISM inhibited platelet aggregation and serotonin release induced by either ADP or epinephrine in a dose dependent manner. This effect of ISM was observed with both gel-filtered platelets (ID50 = 8-30 micrograms ISM/ml gel-filtered platelets) and platelets in plasma (ID50 = 400-900 micrograms ISM/ml of platelet-rich plasma). The active molecule(s) in ISM was heat stable, resistant to acid, base and proteolysis and fractionated on Sephadex 6-25 at MW approximately 280. ISM did not interact with the platelet alpha-adrenergic receptor, but increased cAMP in intact platelets. The results are consistent with the concept that ISM inhibition of platelet aggregation and release is mediated by an increase in platelet cAMP. The exact mechanism whereby ISM increases platelet cAMP appears to be that of inhibition of cyclic AMP phosphodiesterase. The effect of ISM on platelet function is one mechanism which might explain the therapeutic effect of ISM in experimental and clinical coronary artery disease.

Adenylyl Cyclases↗
PubMed1982

Factor V binding to human platelets: the role of divalent cations and of platelet preparatory methods.

The binding of bovine factor V to human platelets has been studied to ascertain the influence of divalent cations as well as the manner of preparation of platelet suspensions. The binding of factor V to platelets was inhibited by EDTA and EGTA and required calcium (optimum concentration 5mM) but not magnesium. Factor V bound to a single class of low affinity sites in unstimulated gel filtered platelets with a Kd of 3.42 x 10(-8)M. Approximately, 2,340 factor V molecules were bound per platelet. Stimulation with the calcium ionophore A23187 or ADP and fibrinogen and inhibition with prostaglandins E1 (PGE1) or prostaglandins I2 (PGI2) failed to alter these constants. However, washing platelets by repeated centrifugation even in the presence of inhibitors of platelet activation decreased the number of binding sites to 1200 without change in the kd. These studies demonstrate a requirement for calcium in the physiological range for factor V binding to platelets, establish that washed platelets bind fewer factor V molecules than gel filtered platelets, and indicate that stimulation or inhibition of platelet function does not affect factor V binding.

Adenosine Diphosphate↗
PubMed1982

Coagulation factor V exists uncomplexed in bovine plasma.

Bovine coagulation factor V has been examined immunochemically to ascertain whether the coagulant polypeptide (h) with Mr = 290,000-330,000 is complexed in plasma with a second immunochemically distinct polypeptide (l2) of Mr = 400,000. Antiserum containing antibodies to h and l2 detects the l2 polypeptide eluting earlier than the h chain on gel filtration of plasma with either added calcium of EDTA, consistent with the behavior of a higher molecular weight noninteracting species. An immobilized monospecific antibody to l2 removes only the l2 polypeptide from a purified factor V preparation containing both h an l2. Moreover, while a monospecific antibody to the h chain was able to precipitate purified radioactively labelled h chain alone or mixed with plasma, the l2 antibody was unable to precipitate radioactively labelled h chain even after attempted recombination of the h chain with l2 present in plasma. These studies indicate that the l2 polypeptide is not complexed to the h chain in a purified system or in plasma and reinforce the conclusion that factor V is a single polypeptide chain uncomplexed in plasma.

Animals↗