Interaction of nucleotide affinity analog 5'-p-fluorosulfonylbenzoyladenosine with platelet ADP receptor: aggregin.
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Adenosine↗
Biomedical subjects
R W Colman
Publications and source records attributed to R W Colman.
Role of kinins in pain and hyperalgesia: psychophysical studies in a patient with kininogen deficiency.
1. Bradykinin is considered to be an important mediator of pain and hyperalgesia associated with injury and inflammation. Psychophysical studies were conducted in a patient with complete kininogen deficiency to determine whether the absence of bradykinin was associated with abnormalities in pain sensibility. Pain evoked by heat stimuli to the thenar eminence was tested before and after a localized burn, which has been shown to cause hyperalgesia in normal subjects. In addition, pain evoked by intradermal administration of bradykinin (0.1-10 micrograms) to the forearm and the effects of bradykinin on pain induced by heat stimuli were studied. The patient rated the intensity of pain evoked by all heat stimuli relative to the pain induced by a 3 s 45 degrees C stimulus. 2. The patient's heat pain threshold (45 degrees C) in the glabrous skin was similar to that of age-matched control subjects (n = 5) and to that previously observed in younger control subjects. 3. The burn resulted in a decrease in pain threshold and an increase in pain induced by suprathreshold stimuli. The magnitude of hyperalgesia was within the range observed in the age-matched control subjects and in younger control subjects. Thus, kinins are not essential for the development of hyperalgesia after heat injury. 4. In control subjects, intradermal injections of bradykinin produced pain and hyperalgesia to heat stimuli. In the patient, intradermal bradykinin injections induced minimal pain and no hyperalgesia to heat stimuli. Thus, congenital absence of kininogens may be associated with a deficiency in bradykinin receptors.
Aged↗
Chromosomal mapping of human kininogen gene (KNG) to 3q26----qter.
The structural gene for human kininogen (KNG) was localized to chromosome 3q26----qter by in situ hybridization. The assignment substantiates the evolutionary relationship of kininogen to two other members of the cystatin superfamily, alpha-2-HS-glycoprotein and histidine-rich glycoprotein, which also map to chromosome 3.
Blood Proteins↗
Clopidogrel inhibits the binding of ADP analogues to the receptor mediating inhibition of platelet adenylate cyclase.
Clopidogrel, like the homologous thienopyridine derivative ticlopidine, selectively inhibits platelet aggregation induced by ADP. We have previously described two nucleotide-binding sites on platelets related to ADP-mediated platelet responses. The first is a high-affinity binding site for 2-methylthio-ADP (2-MeSADP) that is linked to the inhibition of stimulated adenylate cyclase. The second is the 100-kd exofacial membrane protein aggregin, which is labeled by the reactive ADP analogue 5'-p-fluorosulfonylbenzoyl adenosine (FSBA) that is related to shape change and aggregation. We set out to determine if either of these sites is blocked in vivo by clopidogrel or its active metabolite. Six subjects were given clopidogrel (75 mg/day for 10 days) in a double-blind crossover experiment. All of the subjects developed prolonged bleeding times while taking the drug. The rate of onset of the effect on bleeding time varied among subjects. Platelet aggregation induced by ADP or thrombin was significantly impaired by the drug treatment, but no effect was detected on shape change. The incorporation of [3H]FSBA into aggregin was also unaffected. Inhibition of adenylate cyclase by ADP or by 2-MeSADP was greatly reduced in all subjects, and in the case of 2-MeSADP, there was evidence for a noncompetitive effect. Inhibition of adenylate cyclase by epinephrine was unaffected. In the three subjects for whom binding measurements were made, the number of binding sites for [32P]2-MeSADP was reduced from 534 +/- 44 molecules per platelet during control and placebo periods (11 determinations) to 199 +/- 78 molecules per platelet during drug treatment (three determinations). There was no consistent change in the binding affinity.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine↗
Disseminated intravascular coagulation. Approach to treatment.
Disseminated intravascular coagulation (DIC) is a syndrome caused by the systemic generation of thrombin. Most cases are due to pathological activation of the intrinsic coagulation systems (e.g. in sepsis), and/or the extrinsic system (e.g. in malignancy and head trauma). Diagnosis is made by finding abnormalities in at least 3 of 4 laboratory values, namely prothrombin time, platelet count, fibrinogen and fibrinogen/fibrin degradation products. The most common clinical manifestation of DIC is bleeding, with thrombosis in less than 10% of acute cases but more frequently encountered in chronic DIC associated with malignancy. Acute DIC must first be treated by specific therapy of the underlying disease and general support measures. If serial clinical and laboratory monitoring improves, no further treatment is required. If severe or life-threatening haemorrhage occurs or a thrombotic event ensues, heparin anticoagulation followed by aggressive replacement with platelets, fresh plasma and possibly cryoprecipitate is indicated. Heparin doses should be 'therapeutic' (i.e. adequate to overcome the coagulant forces that may have produced a relative heparin-resistant state in the blood). Chronic DIC with haemorrhage, or more usually thrombosis, should also be treated with heparin; warfarin is ineffective. If DIC persists because, for example, a tumour does not regress, long term outpatient subcutaneous heparin therapy may be required.
Disseminated Intravascular Coagulation↗
Role of kallikrein-kinin system in the pathogenesis of bacterial cell wall-induced inflammation and enterocolitis.
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Animals↗
Low-Km cyclic AMP phosphodiesterase from human platelets. Stimulation of activity by phosphorylation of the enzyme and affinity labeling of the active site.
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3',5'-Cyclic-AMP Phosphodiesterases↗
Activation of the contact system in lethal hypotensive bacteremia in a baboon model.
The hypotension in septicemia is believed to be mediated by the combined action of many mediators including cytokines, prostaglandins, and complement components. To evaluate the contribution of the contact/kinin-forming system to hypotension, the authors used an established experimental baboon model of bacteremia in which two concentrations of Escherichia Coli (E. coli) were used to produce lethal and nonlethal hypotension. The lethal group (n = 5) developed irreversible hypotension that significantly correlated with the decline in levels of high molecular weight kininogen (HK) and an increase in alpha 2 macroglobulin-kallikrein complexes (alpha 2M-kal). The nonlethal group (n = 9) experienced reversible hypotension, a less striking decline in HK, and only slight elevation in alpha 2M-kal. No significant changes were found in levels of factor XII, prekallikrein, and factor XI in either group. A significant change in the contact system, which reflects the fatal outcome, is the rise in alpha 2M-kal. This study suggests that irreversible hypotension correlates with prolonged activation of the contact system.
Animals↗
The contribution of factor V to the coagulant property of pleural fluid.
Intrapleural fibrin deposition commonly accompanies pleural injury and may contribute to the organization of exudative pleural effusions, which leads to lung entrapment. Previous investigators have observed an increase in procoagulant proteins in pleural effusions but very little thrombin formation. FVa is the protein cofactor in the prothrombinase complex that dramatically enhances the generation of thrombin from prothrombin by the serine protease fXa. The presence of fVa within the pleural space could influence fibrin formation and pleural scarification. We examined pleural fluids obtained from patients who had lung cancer, CHF, and empyema for the presence of fV/fVa. The fV antigen was increased in exudative pleural fluids, in comparison with transudates. However, the specific activity of fV antigen present in exudates was significantly less than that observed for the lower concentration of antigen present in transudate and could not be activated to the same degree by thrombin. Immunoblots of fV antigen in exudates indicated that fV was partially cleaved and inactivated by unidentified proteases. We conclude that although fV is present in pleural fluid, it may be present in a degraded form, which may partially account for a lack of thrombin-generating capacity in these pleural fluids. The presence of fV does not necessarily correlate with pleural loculation.
Blood Coagulation Factors↗
Sensitive antigenic determinations of high molecular weight kininogen performed by covalent coupling of capture antibody.
Knowledge of the role of high-molecular-weight kininogen (HK) in disease states has been limited by the lack of easy, reliable, and rapid assays for HK antigen. We have developed two immunochemical determinations, which use covalent coupling of the capture antibody, that will greatly facilitate the measurement of HK in plasma and cells. The first is an enzyme-linked immunosorbent assay (ELISA) that is performed in 96-well microplates and can be prepared up to 5 months in advance; it can, therefore, be available when needed. The second is a very rapid method that uses the technique known as particle concentration fluorescence immunoassay (PCFIA) and can be performed on 96 samples in less than 30 minutes. The HK-ELISA correlated well with HK coagulant activity in 22 normal donors (r = 0.88). The interassay coefficient of variation (CV) of 22 samples assayed on 5 separate days was 12%, whereas the interassay CV was 4%. The HK-PCFIA demonstrated an excellent correlation (r = 0.97) with HK coagulant activity in 22 normal donors, with an interassay CV of 7% and an intraassay CV of 1.5%. The HK ELISA was linear between 6 and 80 ng/ml, whereas the HK-PCFIA was linear between 5 and 800 ng/ml. Using the HK-PCFIA, we found a difference in HK antigen levels between two groups of patients with sepsis. The mean of those who survived (n = 10) was 70 micrograms/ml, whereas the mean of those who died (n = 6) was 48 micrograms/ml. Both HK-ELISA and HK-PCFIA were able to detect HK antigen in baboon plasma, enabling the monitoring of HK during experimental protocols. The HK-PCFIA was sensitive enough to detect HK antigen in the supernatant of hepatoma G2 at levels of 1 to 4 ng/ml. These assays should enable convenient and frequent measurement of HK antigen in various physiologic and pathophysiologic states, as well as in cell fractions.
Animals↗
Plasma elastase alpha 1-antitrypsin and lactoferrin in sepsis: evidence for neutrophils as mediators in fatal sepsis.
Increased vasopermeability and vasodilation, presumably the result of endothelial perturbation, are considered among the basic pathogenetic mechanisms in septic shock. Neutrophils have been implicated as a source for mediators in endothelial injury. We measured elastase-alpha 1-antitrypsin (alpha 1AT) complexes and lactoferrin as markers for release of neutrophil granule contents in plasma from patients with sepsis on admission to the Intensive Care Unit, and we delineated the relationship of neutrophil activation to other inflammatory parameters and to hemodynamic and biochemical parameters. Levels of elastase-alpha 1AT and lactoferrin significantly correlated with each other (r = 0.58; p less than 0.008), and were increased (greater than 3.33 and 5 nmol/L, respectively) in 96% and 71% of the patients, respectively. Lactoferrin, but not elastase-alpha 1AT, correlated with the number of white blood cells (r = 0.38; p = 0.008). Elastase-alpha 1 AT levels were significantly higher (p = 0.008), whereas white blood cell counts were lower (p = 0.015) in patients with shock when compared with patients without abnormal blood pressure. Both elastase-alpha 1AT and lactoferrin levels correlated with lactate levels (r = 0.33; p = 0.024 and r = 0.30; p = 0.04), suggesting a role for neutrophil activation in the pathogenesis of hypoxygenation. In addition, elastase-alpha 1AT correlated with the concentrations of interleukin 6 (IL-6) (r = 0.46; p = 0.001) and C3a (r = 0.38; p = 0.009), suggesting that cytokines and complement may contribute to the degranulation of neutrophils in sepsis. Elastase-alpha 1AT complexes were inversely related to C1-inhibitor (r = -0.33; p = 0.028) and to platelet numbers (r = -0.42; p = 0.003). Levels of elastase-alpha 1AT complexes in plasma appeared to be of prognostic significance; levels were higher in 27 patients who died than in 21 patients who survived (p = 0.01). The mortality in 27 patients with concentrations below 10 nM was 37%, whereas it was 81% in 21 patients with higher levels. The overall mortality in this study was 56%. These results provide further evidence that activation and degranulation of neutrophils, induced by multiple agonists, are involved in the development of fatal complications in patients with sepsis.
Bacterial Infections↗
Effect of heparin on the activation of factor XII and the contact system in plasma.
We examined in purified systems and in human plasma whether heparin serves as a contact system activating compound. Purified human factor XII zymogen was not activated by heparin through an autoactivation mechanism, but was activated in the presence of purified prekallikrein. Zn2+ (12 microM) did not support autoactivation by heparin. The activation of factor XII and the contact system by heparin in plasma anticoagulated with citrate or with hirudin (not chelating ions) was examined by the cleavage of 125I-labeled factor XII and high molecular weight kininogen (HK). Heparin at 1.6 and 16 USP U/ml was not able to produce activation, in contrast to dextran sulfate (20 micrograms/ml) which supported activation of both factor XII and HK. This study indicates that heparinized plasma does not support activation of the contact system mediated through activation of factor XII. It is not expected that heparin anticoagulant therapy will contribute to activation of the contact system.
Complement C1 Inactivator Proteins↗
Alpha 2-macroglobulin-kallikrein complexes detect contact system activation in hereditary angioedema and human sepsis.
Activation of the contact system has been documented in severe sepsis and hereditary angioedema, but a sensitive, specific, and quantitative assay for assessing the degree of involvement of this proteolytic enzyme cascade is not yet available. We have developed a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) for the alpha 2-macroglobulin-kallikrein (alpha 2M-Kal) complex using an F(ab')2 derivative of a monospecific polyclonal antibody against alpha 2 M as the capture antibody and a unique murine monoclonal antibody, 13G11, against the heavy chain of kallikrein as the detector antibody. The assay does not detect complexes in normal plasma but reacts with complexes generated by activating normal plasma with dextran sulfate at 4 degrees C in a range of 5 to 375 nmol/L. A close correlation of the ELISA with an amidolytic assay for alpha 2M-Kal was documented. Patients with sepsis syndrome but negative bacterial blood cultures did not show elevated plasma complexes, whereas a majority of those with positive blood cultures did show modest elevation and a single patient with septic shock showed a very high level of alpha 2M-Kal complex. Similarly, a patient with classic hereditary angioedema (HAE) showed increased concentration of complexes on three separate occasions during attacks but normal levels between attacks. Two other HAE patients did not show elevated levels at quiescent periods. The ELISA for alpha 2M-Kal appears to be sensitive, specific, and quantitative, and it can be used to reflect the degree of contact system activation in human sepsis and in HAE attacks.
Angioedema↗
Localization of distinct functional domains on prekallikrein for interaction with both high molecular weight kininogen and activated factor XII in a 28-kDa fragment (amino acids 141-371).
The predominant autolytic form of human kallikrein, beta-kallikrein, was used to localize the high molecular weight kininogen (HK) binding site on kallikrein as well as the substrate recognition site for activated factor XII on prekallikrein. beta-Kallikrein is formed by autolysis of the kallikrein heavy chain to give two fragments of approximately 18 and 28 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A ligand binding technique established that the HK binding site on kallikrein residues on the 28-kDa fragment of the heavy chain. Limited NH2-terminal sequencing of this portion of beta-kallikrein showed that this fragment of the heavy chain consists of the COOH-terminal 231 amino acids of the heavy chain. A panel of five murine monoclonal antibodies to human prekallikrein (PK) were found to have epitopes on this same fragment of the heavy chain. None of the monoclonal antibodies were able to block binding of HK to PK. Three of the monoclonal antibodies (13G11, 13H11, and 6A6) were able to inhibit the activation of PK to kallikrein in both a plasma system and a purified system. The 28-kDa fragment of the PK heavy chain was purified and was able to compete with HK for binding to PK. The HK binding site and the site of recognition of factor XII are separate and distinct on PK, and both are contained in the COOH-terminal 231 amino acids of the PK heavy chain.
Animals↗
Inhibition of ADP-induced platelet shape change and aggregation by o-phthalaldehyde: evidence for covalent modification of cysteine and lysine residues.
Platelets play a major role in the hemostatic process following vascular injury. Chemical modification of cysteine and/or lysine residues in platelet proteins has been shown to cause loss of platelet aggregation induced by diverse agonists; however, these investigations have not addressed the identity of the specific proteins affected. o-Phthalaldehyde (OPTH) is a unique chemical modification reagent that forms and permits the identification of fluorescent isoindole derivatives with proteins by covalently and simultaneously modifying closely spaced cysteine and lysine residues. We found that OPTH inhibited platelet aggregation induced by ADP, collagen, and U46619 (an analog of prostaglandin H2), but had minimal effect on platelet aggregation induced by thrombin, plasmin, chymotrypsin, A23187 (a calcium ionophore), PMA (phorbol 12-myristate 13-acetate), and PMA + A23187. Since platelet aggregation induced by ADP, collagen, and U46619 has been shown to involve binding of endogenous or exogenous ADP to the platelet receptor, our further studies focused on platelet aggregation induced by ADP. OPTH inhibited ADP-induced shape change and aggregation in a concentration-dependent manner. The second-order rate constant for the inhibition of ADP-induced platelet shape change (Ksc = 1.0 X 10(3) M-1 s-1) was lower than that for aggregation (Kagg = 5.4 X 10(3) M-1 s-1). Fluorescence excitation and emission spectra of OPTH-platelet adduct exhibited maxima at 346 and 437 nm, respectively, consistent with the formation of an isoindole derivative(s). The nonpenetrating thiol-specific reagent, p-chloromercuribenzenesulfonate (pCMBS) (0.8 mM), is known to block the inhibition of stimulated adenylate cyclase induced by ADP but not the ADP-induced platelet shape change. The inhibition of ADP-induced platelet shape change (Ksc = 1.5 X 10(3) M-1 s-1) by OPTH was not affected by pCMBS. OPTH, at concentrations (15-50 microM) that inhibited ADP-induced platelet aggregation and shape change did not raise the intracellular levels of adenosine cyclic 3',5'-monophosphate (cAMP) in platelets nor did it impair the ability of iloprost (a stable analog of prostaglandin I2) to raise the platelet cAMP level. Thus, OPTH under these conditions did not interact with platelet adenylate cyclase. 5'-p-fluorosulfonylbenzoyladenosine (FSBA) has been previously shown to inhibit ADP-induced platelet shape change and aggregation by covalently modifying aggregin (Mr = 100 kDa), a putative ADP receptor on platelet surface.(ABSTRACT TRUNCATED AT 400 WORDS)
Adenosine↗
Western blot analyses of prekallikrein and its activation products in human plasma.
Prekallikrein (PK), a zymogen of the contact system, and its activation products, kallikrein (KAL), KAL-inhibitor complexes and fragments containing KAL epitope(s) have been detected in human plasma by immunoblotting with a monoclonal anti-human plasma PK antibody, MAb 13G11. Detection of antigen-MAb 13G11 complexes with peroxidase-conjugated anti-IgG showed that the two variants of PK (85- and 88-kDa) are the only major antigen species in normal, non-activated plasma. Upon plasma activation with kaolin, the intensity of the PK bands decreased with formation of complexes of KAL with C1 inhibitor (C1 INH) and alpha 2-macroglobulin (alpha 2 M) identical to those formed by the purified proteins. Immunoblots of normal plasma showed good correlation between the PK detected and the amount of plasma assayed. Increasing amounts of KAL incubated with a constant volume of PK-deficient plasma showed increasing amounts of KAL and of KAL-C1 INH and KAL-alpha 2 M complexes. Complexes of KAL-antithrombin III (ATIII) and the ratio of KAL-alpha 2 M/KAL-C1 INH were higher in activated C1 INH-deficient plasmas than in activated normal plasmas. Protein resolution by 3-12% gradient SDS-PAGE and epitope detection with [125I]MAb 13G11 showed four KAL-alpha 2 M species and a 45-kDa fragment(s) in both surface-activated normal plasma and complexes formed by purified KAL and alpha 2 M. Immunoblots of activated plasma also showed bands at the position of KAL-C1 INH and KAL-ATIII complexes. When alpha 1-antitrypsin Pittsburgh (alpha 1-AT. Pitts) was added to plasma before activation, KAL-alpha 1-AT. Pitts was the main complex.(ABSTRACT TRUNCATED AT 250 WORDS)
Antibodies, Monoclonal↗
High molecular weight kininogen inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets.
In this study we show that high molecular weight kininogen (HK) inhibited alpha-thrombin-induced aggregation of human platelets in a dose-dependent manner with complete inhibition occurring at plasma concentration (0.67 mumol/L) of HK. HK (0.67 mumol/L) also completely inhibited thrombin-induced cleavage of aggregin (Mr = 100 Kd), a surface membrane protein that mediates adenosine diphosphate (ADP)-induced shape change, aggregation, and fibrinogen binding. The inhibition of HK was specific for alpha- and gamma-thrombin-induced platelet aggregation, because HK did not inhibit platelet aggregation induced by ADP, collagen, calcium ionophore (A23187), phorbol myristate acetate (PMA), PMA + A23187, or 9,11-methano derivative of prostaglandin H2 (U46619). These effects were explained by the ability of HK, at physiologic concentration, to completely inhibit binding of 125I-alpha-thrombin to washed platelets. As a result of this action of HK, this plasma protein also completely inhibited thrombin-induced secretion of adenosine triphosphate, blocked intracellular rise in Ca2+ in platelets exposed to alpha- and gamma-thrombin, inhibited thrombin-induced platelet shape change, and blocked the ability of thrombin to antagonize the increase in intracellular cyclic adenosine monophosphate (cAMP) levels induced by iloprost. Because elevation of cAMP is known to inhibit binding of thrombin to platelets, we established that HK did not increase the intracellular concentration of platelet cAMP. Finally, HK did not inhibit enzymatic activity of thrombin. To study the role of HK in the plasma environment, we used gamma-thrombin to avoid fibrin formation by alpha-thrombin. Platelet aggregation induced by gamma-thrombin was also inhibited by HK in a dose-dependent manner. The EC50 (concentration to produce 50% of the maximum rate of aggregation) of gamma-thrombin for washed platelets was 7 nmol/L and increased to 102 nmol/L when platelets were suspended in normal human plasma. The EC50 for platelet aggregation induced by alpha-thrombin in plasma deficient in total kininogen was 40 nmol/L. When supplemented with HK at plasma concentration (0.67 mumol/L), the EC50 increased to 90 nmol/L, a value similar to that for normal human plasma. These results indicate that (1) HK inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets; (2) HK is a specific inhibitor of platelet aggregation induced by alpha- and gamma-thrombin; and (3) HK plays a role in modulating platelet aggregation stimulated by alpha-thrombin in plasma.
Adenosine Triphosphate↗
Structure and functions of human kininogens.
Evidence has accumulated over the past three decades implicating plasma kininogens in numerous inflammatory processes. Delineation of the detailed biochemistry and, more recently, the molecular biology of the human kininogens has resulted in a deeper understanding of the structure-function correlations of the human kininogens. Studies of alterations of human kininogens in disease states have yielded information about the mechanisms of their involvement in inflammatory states. Here, Raul DeLa Cadena and Robert Colman summarize kininogen function in relation to structure and diagnostic and therapeutic potential.
Humans↗