Biomedical subjects

R W Colman

Publications and source records attributed to R W Colman.

At least 73 records · Page 4Linked to original sources

PubMed1997

Human kininogens regulate thrombin binding to platelets through the glycoprotein Ib-IX-V complex.

We and others have shown that both high and low molecular mass kininogens are able to inhibit the thrombin-induced aggregation of gel-filtered platelets, indicating that the locus for inhibition resides in the heavy chain. The inhibitory site is present in domain 3, confined to the C-terminal portion of the region encoded by exon 7 (K270-G292), and the minimal effective sequence is a heptapeptide (L271-A277; Kunapuli et al, J Biol Chem 271:11228, 1996). Kininogens inhibit thrombin binding to platelets and thus inhibit thrombin-induced aggregation. The molecular mechanism by which kininogens inhibit thrombin-induced aggregation of platelets is unknown. Thrombin has previously been shown to bind to two receptors on the platelet surface, glycoprotein (GP) Ib-IX-V complex and the hepta-spanning transmembrane receptor coupled to G protein(s). We now show that, unlike its effect on normal platelets, kininogen (2 micromol/L) did not inhibit the thrombin-induced aggregation of Bernard-Soulier platelets, which lack the GP Ib-IX-V complex, suggesting that kininogen interacts either directly or indirectly with that complex and restricts access by thrombin to this receptor. We further show that both recombinant K270-G292 polypeptide and the synthetic peptide L271-A277 derived from high molecular mass kininogen lower thrombin binding to platelets in a manner similar to monoclonal antibodies to or ligands (von Willebrand factor and echicetin) of GP Ib-IX. The anti-GP Ib-IX-V complex antibodies, TM-60 and SZ 2, can inhibit 125I-high molecular mass kininogen binding to platelets. Conversely, kininogen could block the binding of biotinylated TM-60 or of 125I-SZ 2. Kininogen inhibited the binding of biotinylated thrombin bound to a mouse fibroblast cell line transfected with the GP Ib-IX-V complex. These results indicated that kininogen binds to the GP Ib-IX-V complex modulating thrombin binding to platelets and the consequent platelet aggregation. Kininogen can thus serve as an important regulator of the early stages of platelet stimulation by thrombin.

Animals↗

PubMed1997

High molecular weight kininogen peptides inhibit the formation of kallikrein on endothelial cell surfaces and subsequent urokinase-dependent plasmin formation.

A sequence of 31 amino acids (S565-K595) in domain 6 of the light chain of high molecular weight kininogen (HK) has previously been shown to be responsible for the binding of plasma prekallikrein (PK) or kallikrein. To find effective peptides that might block binding between HK and PK on cell surfaces, a new series of synthetic peptides has now been prepared that incorporates portions of this binding domain sequence. For mapping the minimal sequence within HK, these new peptides were tested for their ability to compete with HK for binding PK in a cell-free system and on human umbilical vein endothelial cells (HUVEC). In the former, at pH 7.4, the kds for binding between kallikrein and either D567-K595, S565-P594, D567-S593, or D567-T591 were all similar to that for the binding of S565-K595 (0.2 to 0.4 micromol/L), but those for the binding of D568-K595, W569-K595, and D567-P589 were an order of magnitude greater (kd = 2 to 5 micromol/L). D567-S586, the shortest chain length of the N- and C-terminal truncation sequences tested, does not effectively compete with kininogen for kallikrein binding (kd = 100 micromol/L). These results imply that D567-T591, a 25-residue peptide (HK25c), contains sufficient structural information for binding kallikrein in solution. D567-T591 also is the minimum structural sequence to block binding of kallikrein to HUVEC-bound HK (IC50 = 50 nmol/L) and to inhibit PK activation to kallikrein on the cell surface (IC50 = 80 nmol/ L). In addition, D567-T591 also inhibits the generation of kallikrein-activated urokinase, which activates plasminogen to plasmin (IC50 = 100 nmol/L). Thus, HK-derived peptides may be useful compounds for modulating excessive fibrinolysis and hypotension in sepsis and multiple trauma.

Amino Acid Sequence↗

PubMed1997

Modulation of platelet responses by 2-[3-(bromo-2-oxopropylthio)]adenosine-5'-diphosphate involves its binding to as well as covalent modification of an ADP-receptor, aggregin.

The 2-substituted ADP derivatives are known to activate human blood platelets with varying degrees of potency. For example, 2-(4-bromo-2,3-dioxobutylthio)adenosine-5'-diphosphate [2-BDB-TADP], an ADP-affinity analog, was previously shown by us to be 50% as potent as ADP in inducing human blood platelet responses [Puri, R. N., Colman, R. F., and Colman, R. W. (1996) Eur. J. Biochem. 236, 862-870]. 2-Methylthio-ADP (2-MeS-ADP) has been known to be a far more potent agonist than ADP. However, the molecular basis for defining the rank order of potency of the 2-substituted ADP derivatives as agonists of platelet responses have been incompletely understood. We now report that 2-BOP-TADP (a one carbon atom lower homolog of 2-BDB-TADP) at equimolar concentration is as potent as ADP in inducing platelet responses. Prolonged incubation of platelets with 2-BOP-TADP abolished its ability to elicit cellular responses. An autoradiogram of the gel obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized platelets labeled by incubating the platelets with 2-BOP-TADP for 1 h followed by reduction by NaB[3H]4 showed the presence of a single covalently radiolabeled protein band at 100 kDa. Preincubation of platelets with either ADP or ATP reduced the intensity of the band corresponding to the 100-kDa protein radiolabeled by 2-BOP-TADP and NaB[3H]4. The results show that (i) 2-BOP-TADP modulates ADP-induced platelet responses by interacting with aggregin and (ii) 2-BOP-TADP was twice as potent as 2-BDB-TADP, and (iii) the chain length of the substituent in a homologous series has an important bearing on the potency of a 2-substituted ADP analog.

Adenosine Diphosphate↗

PubMed1997

Inactivation of recombinant monocyte cAMP-specific phosphodiesterase by cAMP analog, 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate.

Two cAMP analogs, 8- and 2- [(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate (8- and 2-BDB-TcAMP) have been used in probing the catalytic site of recombinant monocyte cAMP-specific phosphodiesterase (PDE4a). 2-BDB-TcAMP is a reversible and competitive inhibitor (Ki = 5.5 mumol/L) of cAMP hydrolysis by PDE4a, 8-BDB-TcAMP irreversibly inactivates the enzyme in a time- and concentration-dependent manner with a second order rate constant of 0.022 mmol/L-1 min-1. The rate of inactivation of PDE4a is reduced by the presence of the substrate cAMP and specific inhibitors, rolipram and denbufylline, but not by cGMP or AMP. Reduction of the enzyme-inhibitor complex with sodium [3H]borohydride shows that 1.2 mol of the affinity label/mol of enzyme was incorporated. The radiolabeled peptide is composed of 10 amino acid residues (697 to 706) located near the carboxyl end of the proposed catalytic domain. The peptide (GPGHPPLPDK) has seven nonpolar and aliphatic residues, of which four are proline, giving the peptide a highly structured conformation. This peptide is the first to be identified in the putative catalytic domain involved in substrate recognition.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1997

Involvement of endogenous kinins in the pathogenesis of peptidoglycan-induced arthritis in the Lewis rat.

OBJECTIVE: To investigate the pathophysiologic roles of endogenous bradykinin (BK) and des-Arg9-BK on local and systemic inflammatory responses in a rat model of acute arthritis induced by peptidoglycan-polysaccharide (PG-APS). METHODS: Female Lewis rats were injected intraperitoneally with PG-APS. Selective antagonists of B1 (Lys-[Leu8]-des-Arg9-BK) and B2 (Hoe 140) receptors were infused at 500 microg/kg and 5 mg/kg per day for 6 days, starting 3 days before induction of inflammation, with subcutaneous micro-osmotic pumps. The local inflammatory response was assessed by paw edema, joint swelling, and tissue content of BK and des-Arg9-BK. These peptides were measured by highly sensitive and specific chemiluminescent enzyme immunoassays. Systemic inflammatory reaction was evaluated by the hepatic concentration of the type 2 acute-phase protein T-kininogen. RESULTS: PG-APS induced significant paw edema and joint swelling 24-72 hours after intraperitoneal injection. The maximal responses to PG-APS observed at 72 hours were significantly reduced (31-38%) by the combination of both B1 and B2 receptor antagonists at 5 mg/kg per day. PG-APS induced a significant increase of BK (up to 5.3-fold) and des-Arg9-BK (up to 4.1-fold) 72 hours after challenge. Liver T-kininogen content was increased by 5.3-, 7.7-, and 5.8-fold at 24, 48, and 72 hours, respectively, after PG-APS injection. At 24 hours, Hoe 140 and Lys-[Leu8]-des-Arg9-BK increased liver T-kininogen content by 43% and 45%, respectively, but they had no effect at 72 hours. CONCLUSION: The results indicate that endogenous kinins are involved in local and systemic acute inflammatory responses, through both B1 and B2 kinin receptors, in the model of PG-APS-induced arthritis.

Acute Disease↗

PubMed1997

A baboon model for hematologic studies of cardiopulmonary bypass.

Objective investigation of new inhibitors of blood protein or cellular systems that are activated during cardiopulmonary bypass (CPB) is impeded by the absence of a satisfactory animal model. Because most baboon hematologic proteins immunologically cross-react with those used for human assays, we developed a robust, reusable baboon model of CPB. Blood samples were obtained from adult baboons at six time intervals before, during, and after 60 minutes of partial CPB at 37 degrees C with peripheral cannulas. Both membrane (n = 7) and bubble oxygenators (n = 7) were investigated. We measured platelet and white blood cell counts; platelet response to adenosine diphosphate and release of beta-thromboglobulin; fibrinopeptide A, prothrombin fragment F1.2, thrombin-antithrombin complex, D-dimer, and plasmin-antiplasmin complex; activated complement (C3b/c and C4b/c); elastase-alpha1 proteinase inhibitor complex; and bleeding times. Adherent glycoprotein IIIa antigen in Triton X-100 washes of the perfusion circuit was also measured. Markers of baboon platelet, complement, and neutrophil activation and thrombosis significantly increased during CPB with bubble oxygenator systems but did not change appreciably in membrane oxygenator circuits. Markers of fibrinolysis, D-dimer, and plasmin-antiplasmin complex did not change with either oxygenator. The baboon model of CPB, when a bubble oxygenator is used, is a robust, reusable animal model for evaluating inhibitors of platelet, complement, and neutrophil activation and thrombosis during and after CPB.

Adenosine Diphosphate↗

PubMed1997

Physical and biological significance of peptide sequences mediating the interaction between high molecular weight kininogen and plasma prekallikrein.

HK31 (S565-K595) has previously been shown to encompass the binding domain for plasma prekallikrein (PK) within domain 6 of high molecular weight kininogen (HK). The complementary binding domain for HK within PK is mapped to PK56 (F56-G86), in the Apple 1 domain and to PK266 (K266-C295) in the Apple 4 domain. Isothermal titration calorimetry demonstrated that either PK peptide binds to HK31 in 1:1 stoichiometry. Binding of the alternate PK peptide into a ternary complex is facilitated nearly 2-fold. Fluorescence emission spectroscopy revealed that only the binding of PK56 caused a limited decrease in intrinsic tryptophane fluorescence emission intensity of HK31. We conclude that the two PK peptides bind to the HK peptide at different sites. To map the minimal sequence within HK31, truncated new peptides were tested for their ability to compete with HK for binding PK in a cell-free system. D567-T591, a 25-residue peptide which contains sufficient structural information for binding kallikrein in solution, blocked the binding of kallikrein to HK bound to endothelial cells and inhibited PK activation to kallikrein and the generation of kallikrein-activated urokinase on endothelial cell surfaces. HK-derived peptides could modulate excessive fibrinolysis and hypotension in sepsis and multiple trauma.

Amino Acid Sequence↗

PubMed1997

Activation of plasma contact and coagulation systems and neutrophils in the active phase of ulcerative colitis.

We have shown that the contact (kallikrein-kinin) system is involved in the pathogenesis of experimental enterocolitis. We now investigate activation of the contact and coagulation pathways, platelets, and neutrophils in active and inactive ulcerative colitis patients as compared to normal controls. In active ulcerative colitis patients, a significant decrease of plasma prekallikrein, high molecular weight kininogen, and C1 inhibitor levels was observed as compared with controls, as well as prekallikrein activation on western blots. Significant elevation of prothrombin fragment (F1 + 2), which indicates thrombin generation, and elastase-alpha 1-antitrypsin complexes, reflecting neutrophil activation, were found in patients with active disease. Plasma beta-thromboglobulin, a marker of platelet activation, was elevated in both active and inactive disease and appears to be a feature of ulcerative colitis. Activation of contact and coagulation pathways, as well as neutrophils, may mediate inflammation in the active phase of ulcerative colitis.

Adult↗

PubMed1997

Cyclic AMP phosphodiesterases in human lymphocytes.

The function of lymphocytes, like platelets, has been shown to be inhibited by agents which increase intracellular cyclic AMP. Two high-affinity cAMP phosphodiesterases (PDEs), the cyclic GMP-inhibited cAMP phosphodiesterase, PDE3, and the cAMP-specific phosphodiesterase PDE4, are known to regulate cAMP concentration in haemopoietic cells by degrading cAMP to AMP. We characterized the relative contribution of the two PDEs to total lymphocyte PDE activity. We then determined which of the different gene products, PDE3A, typical of myocardium and platelets, or PDE3B, typical of adipocytes, were present in lymphocytes. The PDE3-specific inhibitor, milrinone, and the PDE4 inhibitor, rolipram, suppressed hydrolysis by 70% and 30% respectively, which indicated that both PDE4 and PDE3 were present, and that PDE3 was predominant. RT-PCR yields the expected size fragment for the primer pair PDE3B and not for PDE3A. The DNA sequence obtained had >95% identity with PDE3B. PDE3B appears to be the major cAMP PDE in lymphocytes. In contrast to human platelets, human lymphocytes appear to contain the PDE3B subtype. Since PDE3B in adipocytes is subject to hormonal regulation, lymphocytes may be similarly modulated. Understanding the role of cAMP regulation and the involvement of cAMP in lymphocyte function may have important implications in drug development.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1997

ADP-induced platelet activation.

Platelet activation is central to the pathogenesis of hemostasis and arterial thrombosis. Platelet aggregation plays a major role in acute coronary artery diseases, myocardial infarction, unstable angina, and stroke. ADP is the first known and an important agonist for platelet aggregation. ADP not only causes primary aggregation of platelets but is also responsible for the secondary aggregation induced by ADP and other agonists. ADP also induces platelet shape change, secretion from storage granules, influx and intracellular mobilization of Ca2+, and inhibition of stimulated adenylyl cyclase activity. The ADP-receptor protein mediating ADP-induced platelet responses has neither been purified nor cloned. Therefore, signal transduction mechanisms underlying ADP-induced platelet responses either remain uncertain or less well understood. Recent contributions from chemists, biochemists, cell biologists, pharmacologists, molecular biologists, and clinical investigators have added considerably to and enhanced our knowledge of ADP-induced platelet responses. Although considerable efforts have been directed toward identifying and cloning the ADP-receptor, these have not been completely successful or without controversy. Considerable progress has been made toward understanding the mechanisms of ADP-induced platelet responses but disagreements persist. New drugs that do not mimic ADP have been found to inhibit fairly selectively ADP-induced platelet activation ex vivo. Drugs that mimic ADP and selectively act at the platelet ADP-receptor have been designed, synthesized, and evaluated for their therapeutic efficacy to block selectively ADP-induced platelet responses. This review examines in detail the developments that have taken place to identify the ADP-receptor protein and to better understand mechanisms underlying ADP-induced platelet responses to develop strategies for designing innovative drugs that block ADP-induced platelet responses by acting selectively at the ADP-receptor and/or by selectively interfering with components of ADP-induced platelet activation mechanisms.

Adenosine Diphosphate↗

PubMed1997

Activation of the kallikrein-kinin system in arthritis and enterocolitis in genetically susceptible rats: modulation by a selective plasma kallikrein inhibitor.

We have developed models of acute and chronic inflammatory arthritis and enterocolitis using peptidoglycan-polysaccharide injected intraperitoneally or subserosally (intramurally) into the distal ileum and cecum. Acute inflammation occurs in both Buffalo and Lewis rats, characterized by inflammation of the injected areas of the intestine. However, only the genetically susceptible Lewis rat develops chronic synovitis and joint erosion or adhesions and granulomatous enterocolitis. In the Lewis rat but not the Buffalo rat, these changes are accompanied by a decrease in plasma prekallikrein and high-molecular-weight kininogen, reflecting activation of the kallikrein-kinin system. Pretreatment with a specific plasma kallikrein inhibitor modulates the acute and chronic arthritis. The same inhibitor partially abrogates the acute changes characteristic of enterocolitis, and preliminary data suggest similar results in the chronic model. The results of these studies indicate that the kallikrein-kinin system plays an important role in arthritis and enterocolitis induced by bacterial products and that kallikrein inhibitors are potential therapeutic agents for inflammatory arthritis and inflammatory bowel disease.

Acute-Phase Proteins↗

PubMed1997

Rapid direct determination of low and high molecular weight kininogen in human plasma by Particle Concentration Fluorescence Immunoassay (PCFIA).

In this study, we employed Particle Concentration Fluorescence Immunoassay (PCFIA), for directly measuring both high and low molecular weight kininogens (HK and LK) in human plasma. In 38 normal donors, the mean values for plasma kininogens were 93 mg/ml +/- 19 SD, 82 mg/ml +/- 12 SD, and 175 mg/ml +/- 29 SD, respectively for LK, HK, and TotK (the sum of LK and HK detected by their common heavy chains). Plasma completely deficient in HK and LK was unreactive (< 0.25 mg/ml) in all 3 assays whereas plasma from a patient with Fitzgerald Trait had an HK value of 11 mg/ml, an LK value of 36 mg/ml and a TotK value of 59 mg/ml. The reagents can be prepared in advance and all three kininogen determinations can be performed, using the same diluted sample on 24 plasma samples, in triplicate, or 40 plasma samples, in duplicate, in less than 1 h. By performing all 3 kininogen determinations, it is possible to differentiate cleaved from intact kininogens. This technique will facilitate the widespread screening of kininogen levels in biological fluids of normal humans as well as of patients with various diseases.

Fluorescent Antibody Technique↗

PubMed1997

Isolation and regulation of the cGMP-inhibited cAMP phosphodiesterase in human erythroleukemia cells.

The predominant cAMP phosphodiesterase in human platelets is the low K(m) cGMP-inhibited phosphodiesterase (PDE 3A). We have isolated native PDE3A from platelets and human erythroleukemia (HEL) cells and studied its kinetics. The platelet and HEL cell enzymes hydrolyze cAMP with a K(m) = 0.5 microM. Incubation of cell supernatant with cAMP dependent protein kinase resulted in a rapid increase in activity within minutes, which resulted from a 2-fold decrease in K(m) with no increase in Vmax. HEL cells grown for 24 h in the presence of 50 microM forskolin, an adenylate cyclase activator, demonstrate further increase in PDE3A of 274% of control (p = 0.03). Cells incubated with forskolin and cycloheximide or actinomycin D demonstrated no increase suggesting that cAMP stimulates PDE3A synthesis by transcriptional regulation. The results indicate that cAMP affects both the short and long-term regulation of PDE3A. The latter effect may play a role in the developing hematopoietic cell and the cardiovascular system to regulate cAMP levels.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1996

Effect of factor Xa inhibitors on thrombin formation and complement and neutrophil activation during in vitro extracorporeal circulation.

BACKGROUND: Even when large doses of heparin are administered during cardiopulmonary bypass, thrombin is produced. Thrombin is a powerful protease that is associated with the thrombotic and bleeding complications of open heart surgery and is produced by cleavage of prothrombin by factor Xa. This study assessed the ability of a specific inhibitor of factor Xa, recombinant tick anticoagulant peptide (rTAP), alone or in combination with standard heparin and a low-molecular-weight heparin, enoxaparin, to suppress thrombin formation and activity during in vitro extracorporeal circulation. METHODS AND RESULTS: Fresh, anticoagulated human blood was recirculated for 2 hours in an extracorporeal membrane oxygenator perfusion circuit at 37 degrees C. Four anticoagulant protocols were evaluated; porcine heparin (3.75 U/mL); enoxaparin (17.5 U/mL); rTAP (4 mumol/L); and porcine heparin plus rTAP (2 mumol/L). Blood samples were obtained for analysis from the donor, after anticoagulation, and after 5, 30, 60, and 120 minutes of recirculation. There were no significant differences between groups in platelet count, response to adenosine diphosphate, or prothrombin fragment (F1.2) production. rTAP plus heparin reduced beta-thromboglobulin release; fibrinopeptide A concentrations were significantly higher with rTAP alone. Enoxaparin strongly and significantly inhibited complement C5b9 production and neutrophil elastase release and was associated with significantly increased concentrations of C1-C1 inhibitor and kallikrein-C1 inhibitor complexes. CONCLUSIONS: rTAP does not reduce thrombin formation or activity during in vitro extracorporeal circulation. Enoxaparin markedly inhibits formation of the complement membrane attack complex and neutrophil elastase release, possibly by accelerating C1 inhibitor activity.

Adenosine Diphosphate↗

PubMed1996

Direct evidence for multifacial contacts between high molecular weight kininogen and plasma prekallikrein.

HK31 (S565-K595) has previously been shown to encompass the binding domain for plasma prekallikrein (PK) within domain 6 of high molecular weight kininogen (HK). The complementary binding domain for HK within PK is mapped to PK56 (F56-G86), in the apple 1 domain, and to PK266 (K266-C295), in the apple 4 domain. Isothermal titration calorimetry was used to directly monitor binding among HK31, PK56, and PK266. Either PK peptide binds to HK31 in 1:1 stoichiometry, regardless of whether a binary complex is first formed between PK266 and HK31 or between PK56 and HK31. Binding of the alternate PK peptide into a ternary complex is facilitated nearly 2-fold. The ternary complex consists of 1:1:1 HK31:PK56:PK266. Furthermore, binary and ternary complex formation is entropically driven and thermodynamically favored, suggesting that the conformational changes accompany binding. Fluorescence emission spectroscopy revealed that binding of PK56 caused a limited decrease in intrinsic tryptophan fluorescence emission intensity of HK31 while binding of PK266 to HK31 or the complex of HK31/PK56 had no such effect. We conclude that the two PK peptides bind to the HK peptide at different sites. The binding between HK and PK is likely due to conformational changes which serve to juxtapose the PK binding domain within HK with the HK binding site involving two spatial proximity segments.

Amino Acid Sequence↗

PubMed1996

A novel method for chemical modification of functional groups other than a carboxyl group in proteins by N-ethyl-5-phenylisooxazolium-3'-sulfonate (Woodward's reagent-K): inhibition of ADP-induced platelet responses involves covalent modification of aggregin, an ADP receptor.

The chemical reaction of N-ethyl-5-phenylisooxazolium-3'-sulfonate (Woodward's Reagent-K, WR-K) with a carboxyl group yields an enol ester that cannot be reduced by sodium borohydride in an aqueous solution, while other nucleophiles such as sulfhydryl, hydroxyl, amino, and imidazole groups, react with WR-K to yield unsaturated ketones that are capable of being reduced by sodium borohydride in an aqueous medium. Aggregin, a 100-kDa protein on the surface of human blood platelets has been identified as an ADP receptor. Autoradiography of the gels obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the samples of solubilized human blood platelets modified by WR-K and then reduced by tritiated sodium borohydride (NaB[3H]4) showed the presence of a prominent band corresponding to a 100-kDa radiolabeled protein. Labeling of platelets by WR-K and NaB[3H]4 was inhibited by ADP, ATP, and thiol group modifying reagents. WR-K blocked completely labeling of platelets by [beta-32P]-8-(4-bromo-2, 3-dioxo-butylthio)adenosine-5'-diphosphate, an ADP-affinity analog that selectively and covalently labels aggregin (Puri, R. N., Kumar, A., Chen, H., Colman, R. F., and Colman, R. W. (1995) J. Biol. Chem. 256, 24482-24488). WR-K also inhibited ADP-induced platelet shape change, aggregation, and mobilization of intracellular Ca2+ and blocked ADP-induced inhibition of stimulated adenylate cyclase activity. The results show conclusively that WR-K inhibited ADP-induced platelet responses by preventing binding of ADP to aggregin and suggest that ADP binding domain of aggregin contains an essential thiol group. The method of labeling proteins by WR-K and NaB[3H]4, hitherto not used to distinguish among functional groups modified by WR-K, offers a useful and convenient alternative to previously used ultraviolet spectral methods which cannot be used to investigate the modified proteins in intact cellular systems.

Adenosine Diphosphate↗

PubMed1996

Human platelet cGI-PDE: expression in yeast and localization of the catalytic domain by deletion mutagenesis.

Cyclic adenosine monophosphate (cAMP) is an important modulator of platelet responses to agonists. Cyclic nucleotide phosphodiesterase (PDE) controls intracellular cAMP concentrations by hydrolyzing it to AMP. The major PDE activity in platelets is PDE3A (cyclic guanosine monophosphate [cGMP]-inhibited PDE). To obtain structural information on platelet PDE3A, we cloned the enzyme cDNA from a human erythroleukemia cell (HEL) library since the cell line expresses many platelet proteins. This clone consists of 87% of the full-length human myocardial PDE3A cDNA, spanning from nucleotides 456 to 4606, and is identical in sequence. The nucleotide coding for the N terminal 179 amino acid sequence (nt 1-536) as well as four other cDNAs (nt 1459-1632, nt 1765-1986, nt 2152-2538, and nt 2978-3375) obtained by RT-PCR of platelet RNA are also identical to the myocardial sequences, indicating that the HEL, myocardial, and platelet PDE3As are the same. Northern blot analysis of HEL cell RNA detected two mRNAs of 7.5 and 4.4 kb. Four new deletion mutants are reported. PDE 3A delta 1 and PDE 3A delta 2, encoding amino acids 665 to 1141 and amino acids 679 to 1141, respectively, were expressed in a PDE-deficient yeast. They displayed PDE activities of 172 and 79 pmol/mg/min, respectively. PDE 3A delta 3 and PDE 3A delta 4, encoding amino acids 686 to 1141 and 700 to 1141, had no detectable PDE activity. All mutant proteins were expressed as determined by Western blot analysis. These findings localize the PDE3A catalytic domain to within amino acid residues 679 to 1141.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1996

Evidence for the presence of essential histidine and cysteine residues in platelet cGMP-inhibited phosphodiesterase.

Camp is a major regulator of platelet function. cGMP-inhibited phosphodiesterase (cGI-PDE) is the predominant platelet enzyme hydrolysing cAMP. The pH-rate profile plot for this enzyme yields pKa values of 6.5 and 9.0, consistent with histidine and cysteine residues respectively. Diethyl pyrocarbonate (DEP) inactivates cGI-PDE in a time- and concentration-dependent manner, and this effect was rapidly reversed by hydroxylamine. It was estimated that 2 mol of histidine residues per mol of enzyme were responsible for the loss of catalytic activity, as deduced from the correlation of the difference spectrum at 240 nm of the DEP-modified cGI-PDE with the enzyme activity. N-Ethylmaleimide (NEM) and 5.5'-dithiobis-(2-nitrobenzoic acid) (DTNB) inactivate cGI-PDE in a time- and concentration-dependent manner, suggesting the selective modification of a cysteine residue. AMP protects the enzyme against DEP, NEM and DTNB, suggesting the presence of histidine and cysteine residues at the active site of cGI-PDE. [14C]DEP incorporation in the presence of AMP or cGMP indicates the protection of two histidine residues by each nucleotide. These residues are different for each agent, since the combination of AMP and cGMP protects four histidine residues. [3H]NEM incorporation showed that 1 mol of cysteine per mol of cGI-PDE was protected by AMP, but not only by cGMP. We conclude that cGI-PDE possesses two essential histidine residues for activity, two additional histidines for cGMP inhibition, and one cysteine residue at the active site.

3',5'-Cyclic-AMP Phosphodiesterases↗