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Biomedical subjects

R W Colman

Publications and source records attributed to R W Colman.

At least 325 records · Page 18Linked to original sources
PubMed1981

Subcellular localization and secretion of factor V from human platelets.

Factor V, a plasma protein cofactor necessary for optimal conversion of prothrombin to thrombin, is also present in considerable concentration in blood platelets (9.9 units per 10(9) platelets). Subcellular fractionation by two methods has localized factor V in the alpha granules of unstimulated platelets. ADP and epinephrine cause release of 4.6% and 6.4%, respectively, of the total factor V, a process completely inhibited by cyclooxygenase alkylation by aspirin. In contrast, collagen causes release of 25% of platelet factor V, a process only partially suppressed by aspirin. Secretion of factor V depends on the availability of metabolic energy, because antimycin A, an inhibitor of aerobic metabolism, and 2-deoxyglucose, an inhibitor of anaerobic glycolysis, together almost totally inhibited the secretion of factor V induced by collagen. The data establish that factor V is not normally available on unstimulated platelets but can be secreted from alpha granules upon stimulation with physiological agents such as ADP, epinephrine, and collagen. Because factor V is known to serve as a receptor for factor Xa, the exposure of factor V on platelets consequent to release would accelerate the process of blood coagulation.

Adenosine Diphosphate↗
PubMed1981

Immunochemical studies of factor V of bovine platelets.

Immunochemical techniques were employed to investigate the molecular properties of bovine platelet factor V. An antiserum prepared against purified factor V rapidly inactivated platelet factor V, indicating that platelet factor V is antigenically related to plasma factor V. A reaction of identity between factor V in plasma and purified factor V was documented by immunodiffusion against an antibody to platelet factor V. When platelet factor V was extracted with Triton X-100 in the presence of protease inhibitors and subjected to immunoelectrophoresis against the antiserum to plasma factor V, a single antigenic component migrating toward the anode was observed. In the absence of protease inhibitors, following release by collagen or solubilization, platelet factor V appeared close to the origin suggesting proteolytic alteration. Platelet factor V released by collagen or extracted with Triton X-100 was activated by thrombin 7.4-fold and 4.5-fold respectively, compared to a 17-fold activation of factor V in plasma under identical conditions. The ability of thrombin to activate platelet factor V as well as the close correspondence of factor V activity and antigen released by collagen indicates that the molecule is largely in the unactivated form after release. A single component of molecular weight 270 000 was seen when platelet factor V released by collagen was immunoprecipitated and subjected to dodecylsulphate gel electrophoresis. Factor V coagulant assays and immunoelectrophoresis of subcellular fractions showed that platelet factor V is localized primarily in the alpha granules. Platelet factor V appears to be similar to, if not identical with, plasma factor V by the criteria of immunologic identity, similar electrophoretic mobility and virtually identical molecular weights.

Animals↗
PubMed1981

Isolation of alpha 1-protease inhibitor from human normal and malignant ovarian tissue.

Proteolytic enzymes are associated with normal and neoplastic tissues. Therefore protease inhibitors might also be involved in the control of cell function. alpha 1-protease antigen and antitryptic activity have been found in normal and neoplastic human ovarian homogenate. The inhibitor has been localized to ovarian stromal cells or tumor cells by immunoperoxidase staining. The protein was purified to apparent homogeneity as judged by alkaline gel and sodium dodecyl sulfate (SDS) gel electrophoresis. Immunochemical studies revealed antigenic similarity of plasma alpha 1-protease inhibitor by double immunodiffusion and similar mobility on immunoelectrophoresis and two-dimensional electroimmunodiffusion. The molecular weight was similar to that described for plasma alpha 1-protease inhibitor: 60,000 by gel filtration and 53,500 by SDS electrophoresis. Furthermore, the phenotypic pattern as determined by acid starch gel electrophoresis and immunoprecipitation was PiMM, which is the predominant genetic variant in normal plasma alpha 1-protease inhibitor. An inhibitor ws isolated and purified from an ovarian carcinoma that exhibited functional, immunochemical, and physical similarity to the normal ovarian alpha 1-protease inhibitor. alpha 1-protease inhibitor from normal and malignant ovaries competitively inhibited bovine pancreatic trypsin at incubation times of 5 min at 30 degrees C. Inhibition constant (Ki) values were calculated at 0.67 and 0.51 inhibitory units, respectively. The alpha 1-protease inhibitor in malignant cells may be a factor in the control of proliferation in this tissue. Since ovulation is in part a proteolytic event, the alpha 1-protease inhibitor in ovarian cells may play a role in the control of this specialized tissue. Persistance of this protein in malignant ovarian tissue may be a vestige of its differentiated origin.

Epitopes↗
PubMed1981

Effect of surfaces on fluid-phase prekallikrein activation.

The activation of prekallikrein by factor XII fragments (XIIf), during incubation in plastic tubes was previously noted to be increased by high molecular weight (HMW) kininogen as well as other plasma proteins. In this report, we investigated the mechanism responsible for this increase. Although we confirmed that HMW kininogen, bovine serum albumin, fibrinogen, cold insoluble globulin, and mixed phospholipids apparently increased prekallikrein activation, we found that the product of prekallikrein activation (kallikrein) lost substantial activity in less than 0.5 min after exposure to a variety of fresh surfaces. This loss was partially prevented by the presence of various proteins and phospholipids. Similar protection against inactivation of XIIf, the enzyme in this reaction, was also found. In contrast, no loss of the substrate, prekallikrein, was observed during incubation. The loss of kallikrein activity was found to be proportional to the surface area of the incubation vessel as well as the concentration of kallikrein. Further loss of kallikrein activity could also be prevented by pretreating the vessel with kallikrein. We therefore conclude that various substances apparently affect prekallikrein activation in a purified system by preventing the enzyme and product in the reaction mixture from losing activity due to adsorption to a surface.

Adsorption↗
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

Persistence of increased platelet cyclic AMP induced by prostaglandin E1 after removal of the hormone.

Prostaglandin E1 (PGE1) covalently linked to omega-NH2-hexyl-agarose (PGE1-hexyl-agarose) stimulates the activity of human platelet adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] 3-fold over control level at 60 sec when stirred at 1200 rpm at 37 degrees C. The time course exhibited a lag phase of 20 sec, a rapid increase to a maximum plateau between 60 and 80 sec, and a more gradual decrease to basal level at about 120 sec. During this entire period of incubation PGE1-hexyl-agarose could be easily separated; therefore it bound only transiently to the platelet. No prostaglandin(s) were found to be released into plasma. The stimulation of adenylate cyclase activity by the insolubilized hormone was dependent on the rate of collision as influenced by the speed of stirring. Removal of the insoluble PGE1 before the end of the lag period (10 sec) prevented the increase of adenylate cyclase. In contrast, separation of PGE1-hexyl-agarose from platelets by filtration after the lag period (at 30 or 40 sec) allowed the stimulation of the enzymatic activity to continue to completion in the absence of the hormone. The results suggest that PGE1 initiates molecular events leading to an increase of adenylate cyclase that do not require its continued presence for maintenance of the stimulated state.

Adenylyl Cyclases↗
PubMed1980

Function and immunochemistry of prekallikrein-high molecular weight kininogen complex in plasma.

Plasma from individuals with high molecular weight (HMW) kininogen deficiency has been reported to be deficient in prekallikrein as measured by radial immunodiffusion, prekallikrein coagulant activity, and/or kaolin-activated arginine esterase activity. The discovery that prekallikrein and HMW kininogen circulate as a complex in plasma led us to reevaluate the antigenic and functional properties of prekallikrein in HMW kininogen-deficient plasma as well as in normal plasma. The low prekallikrein antigen level in an individual with HMW kininogen deficiency was corrected to the normal range (80-95%) by the addition of 0.2 U/ml of purified HMW kininogen. A similar increase in apparent prekallikrein antigen was observed when purified prekallikrein and HMW kininogen were combined. The correction of the apparent prekallikrein defect in this HMW kininogen-deficient plasma coincided with the formation of a prekallikrein-HMW kininogen complex as demonstrated by immunoelectrophoresis. Similar findings were demonstrated with purified prekallikrein and HMW kininogen by immunoelectrophoresis as well as crossed immunoelectrophoresis. The coagulant activity in HMW kininogen-deficient plasma was increased in a dose-dependent manner by the addition of HMW kininogen, reaching 85% of normal level at a concentration of 0.2 U/ml. Kaolin-activated arginine esterase activity (kallikrein) in HMW kininogen-deficient plasma was fully corrected when HMW kininogen was added to the deficient plasma after depletion of kallikrein inhibitors. The functional and antigenic concentration of prekallikrein in plasma from four other HMW kininogen-deficient individuals was similarly corrected to normal after adding HMW kininogen. Addition of HMW kininogen increased the apparent prekallikrein activity in native normal plasma (as measured by esterase activity) but not in normal plasma in which inhibitors were inactivated. The apparent prekallikrein antigen concentration (as measured by radial immunodiffusion or electroimmunodiffusion) increased upon addition of HMW kininogen. Immunoelectrophoresis as well as gel filtration of normal plasma revealed the presence of free prekallikrein (17-38% of the total) in addition to the HMW kininogen-prekallikrein complex previously reported. This study emphasizes the influence of HMW kininogen on both functional and immunologic determinations of prekallikrein.

Antigens↗
PubMed1980

Effect of heterologous antibody on human platelet prostaglandin synthesis.

Rabbit anti-human platelet antiserum (RPA) has been shown to mediate human platelet aggregation and 14C-serotonin release. This effect is not inhibited by aspirin. However, it is not known whether antibody induces platelet prostaglandin synthesis. We, therefore, investigated the effect of RPA on human platelets by measuring malondialdehyde (MDA) production as a reflection of prostaglandin synthesis and comparing it with 14C-serotonin release. Independently, ATP release was also measured as a further measure of platelet secretion. Normal rabbit serum did produce a small amount of MDA (0.54 +/- 0.09 SEM nmole/10(9) platelets). However, RPA produced a much larger amount of MDA (1.43 +/- 0.18 nmole/10(9) platelets) and pre-incubation with aspirin significantly decreased MDA production (0.16 +/- 0.07 nmole/10(9) platelets) (p less than .001). Aspirin did not significantly impair the capacity of RPA to release 14C-serotonin or ATP. These results demonstrate that prostaglandin synthesis occurs when heterologous antibody is added to human platelets. However, prostaglandin formation must be a secondary event in this process since the inhibition of prostaglandin synthesis by aspirin had little effect on the ability of RPA to induce 14C-serotonin release or ATP release.

Adenosine Triphosphate↗
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↗
PubMed1980

Crotalocytin: characterization of the timber rattlesnake platelet activating protein.

Crotalocytin, a platelet activating protein from timber rattlesnake venom, was studied to characterize its nature and to investigate its action on platelets. It exhibited proteolytic activity on the substrate azocoll and amidolytic activity on several peptide p-nitroanilides. The platelet activating and amidolytic activity of Crotalocytin was inhibited by diisopropylfluorophosphate. In addition, phenylmethylsulfonyl fluoride inhibited Crotalocytin's ability to stimulate platelets. Active site titration with p-nitrophenyl guanidobenzoate indicated that 52% of Crotalocytin's molecules were active and that the enzyme could also hydrolyze the titrant. These studies showed that Crotalocytin is a serine protease. Like thrombin and collagen, Crotalocytin induced simultaneous platelet aggregation and adenosine triphosphate (ATP) secretion. EDTA and prostaglandin E (PGE1) blocked Crotalocytin's ability to activate platelets; hirudin and antithrombin III did not. Crotalocytin stimulated the secretion of serotonin from dense granules and low affinity platelet factor 4 and fibrinogen from alpha-granules. Crotalocytin did not cause platelet lactic dehydrogenase loss or agglutinate formalin-fixed platelets, but it did aggregate chymotrypsin-treated platelets. Studies with antimycin A and 2 deoxy- D-glucose showed that Crotalocytin-induced platelet secretion was dependent on metabolic energy. Furthermore, Crotalocytin's induction of platelet secretion was prevented by eliminating exogenous ADP and blocking activation of the arachidonate pathway. Timber rattlesnake venom contains a serine protease that is unique, potent platelet activator.

Adenosine Triphosphate↗
PubMed1980

Identification of two distinct adenosine diphosphate receptors in human platelets.

(1) FSBA, an inhibitor of platelet shape change and aggregation, inhibits and reverses the binding of fibrinogen to washed platelets induced by ADP in the presence of calcium ion; (2) FSBA does not inhibit either aggregation of or fibrinogen binding to platelets treated with chymotrypsin; (3) additional evidence is provided that FSBA does not inhibit aggregation by stimulating cyclic AMP accumulation; (4) FSBA does not inhibit PGE1-induced cyclic AMP accumulation and has little ability to inhibit the action of ADP on cyclic AMP or the binding of the ADP analog [beta 32P] 2-MeSADP; and (5) these results provide additional support for the view that the effects of ADP on aggregation and on cyclic AMP are mediated by distinct receptors, one of which, designated "A", is labeled by FSBA; the second receptor designated "C", has selective affinity for 2-MeSADP.

Adenosine↗
PubMed1980

Collagen-induced platelet shape change. The role of collagen quaternary structure.

Multimerization of collagen molecules appears to be a prerequisite for collagen-induced platelet aggregation and release. Whether a similar requirement exists for collagen-initiated platelet disc-to-sphere transformation, one of the earliest events in the platelet:collagen interaction, is not known. We have studied platelet shape change by highly purified type I collagen in the presence of EDTA to prevent interference from platelet aggregation and release. The ability of soluble collagen to induce platelet shape change was dependent on prior fibril formation. Tropocollagen from which multimers were removed was not able to initiate the disc-to-sphere transformation, indicating that monomeric collagen is insufficient as a platelet activator. Increasing the extent of multimerization by preincubation at 37 degrees led to an increase in the response rate and a decrease in the lag time for initiation of shape change, but even after maximal multimerization, the lag time was not eliminated. The increase in the turbidity of the collagen solution preceded its ability to stimulate platelet shape change, suggesting that a minimum size of the multimer is needed for platelet activation. L-Arginine, an inhibitor of multimerization, prevented collagen-induced shape change in a dose-dependent relationship. Enhancement of the shape change response rate as a function of the concentration of collagen multimers was found.

Animals↗
PubMed1980

Crotalocytin: recognition and purification of a timber rattlesnake platelet aggregating protein.

After being envenomated by the timber rattlesnake, a patient was found to have a platelet count of 5000 per microliter, prothrombin time and activated partial thromboplastin time both greater than 150 sec, plasma fibrinogen 0 mg/dl, and fibrinogen split products 2560 microgram/ml. However, this patient did not appear to have acute disseminated intravascular coagulation since coagulation factors II-XII were normal. We postulated that this venom contained, in addition to a fibrinogen clotting enzyme, a platelet activating protein, Crotalocytin. Crotalocytin was purified from crude timber rattlesnake venom by Sephadex G-100 gel-filtration, low ionic strength precipitation, and DEAE-A50 Sephadex chromatography. By sodium dodecyl sulfate gel electrophoresis and gel-filtration Crotalocytin was a single chain polypeptide, molecular weight 55,000. Thrombocytopenia after timber rattlesnake bite appeared to be due to a protein that directly activated platelets. Timber rattlesnake bite mimicked the clinical presentation of disseminated intravascular coagulation.

Amino Acids↗