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Biomedical subjects

R W Colman

Publications and source records attributed to R W Colman.

At least 181 records · Page 10Linked to original sources
PubMed1991

Role of kallikrein-kinin system in pathogenesis of bacterial cell wall-induced inflammation.

The plasma kallikrein-kinin system is activated in Gram-negative sepsis and typhoid fever, two diseases in which bacterial products have been shown to initiate inflammation. Because a single intraperitoneal injection of bacterial cell wall peptidoglycan-polysaccharide polymers from group A steptococci (PG-APS) into a Lewis rat produces a syndrome of relapsing polyarthritis and anemia, we investigated changes in the role of the kallikrein-kinin system in this model of inflammation. Coagulation studies after injection of PG-APS revealed an immediate and persistent decrease in prekallikrein levels. High-molecular-weight kininogen levels decreased significantly during the acute phase and correlated with the severity of arthritis. Factor XI levels were decreased only during the acute phase. Antithrombin III levels remained unchanged, indicating that neither decreased hepatic synthesis nor disseminated intravascular coagulation caused the decreased plasma contact factors. Plasma T-kininogen (an acute phase protein) was significantly elevated during the chronic phase. PG-APS failed to activate the contact system in vitro. Thus the kallikrein-kinin system plays an important role in this experimental model of inflammation, suggesting that activation of this system may play a role in the pathogenesis of inflammatory bowel disease and rheumatoid arthritis in which bacterial products might be etiologically important.

Animals↗
PubMed1991

Receptors that activate platelets.

This review highlights the increasing knowledge of the biochemistry, pathology, and cell and molecular biology of platelet receptors. A receptor for ADP has been identified using the affinity label FSBA as aggregin, a 100-kDa membrane protein responsible for shape change, aggregation, and exposure of fibrinogen binding sites. A variety of putative receptors for collagen have been described, with GPIa/IIa and GPIV receiving the most attention recently. A thromboxane A2 receptor has been identified using receptor antagonists and photoaffinity labels. The alpha 2-adrenergic receptor has been cloned and expressed. The platelet thrombin receptor has been tentatively identified as GPIb. Following binding of thrombin to this receptor, activation of calpain occurs, with cleavage of aggregin leading to exposure of GPIIb/III alpha and platelet aggregation. Isolation, expression, or both of the ADP, collagen, and thrombin receptors as single gene products of the human platelet responsible for activation, and more complete understanding of stimulus-response coupling, should allow for greater specificity of drugs with selective therapeutic actions.

Animals↗
PubMed1990

Kinetics of inhibition of platelet calpain II by human kininogens.

The plasma kininogens, high-molecular-mass and low-molecular-mass kininogens, are the most potent plasma inhibitors of platelet calpain. We explored the kinetic mechanisms for kininogen inhibition of calpain by comparing calpain inactivation by human high-molecular-mass kininogen (HK) and human low-molecular-mass kininogen (LK). With a [14C]methylated alpha-casein substrate, the inhibition of calpain by HK did not follow classic Michaelis-Menten kinetics. With the use of a fluorogenic assay with the dipeptide substrate for calpain, 3-carboxypropionyl-leucyltyrosine 7-(4-methyl)coumarylamide, the inhibition by HK and LK fitted a kinetic model of tight-binding inhibition. LK was found to be a non-competitive inhibitor of platelet calpain with a Ki of 2.7 nM. HK showed mixed non-competitive inhibition of calpain with a Ki of 2.3 nM in the absence of substrate and Ki of 0.71 nM in the presence of saturating substrate, almost 4-fold tighter than LK. Proteolysis of HK by plasma and tissue kallikreins did not influence its ability to inhibit calpain. Digestion of the HK light chain by Factor XIa also did not alter its calpain-inhibitory function. These studies indicate that the kininogens are tight-binding non-competitive inhibitors of platelet calpain, the inhibitory domain in each case being mainly on the heavy chain. The light chain of HK appears to influence its kinetic behaviour.

Blood Platelets↗
PubMed1990

Purification and characterization of a cyclic GMP-stimulated cyclic nucleotide phosphodiesterase from the cytosol of human platelets.

A cyclic GMP-stimulated cyclic nucleotide phosphodiesterase was purified to near homogeneity from the 150,000 g supernatant fraction of human platelets by a combination of DEAE-cellulose chromatography and cyclic GMP affinity chromatography. Overall purification was about 7400-fold with a 10% to 15% recovery of activity. On NaDodSO4-containing polyacrylamide gels, the purified enzyme migrates as a single band Mr = 105,000. Phosphodiesterase activity co-migrates with the protein band on native polyacrylamide gels. Both Mg2+ and Mn2+ support the activity of this phosphodiesterase. The enzyme hydrolyzes both cyclic AMP and cyclic GMP with similar maximal rates. The hydrolysis of both nucleotides exhibits positive homotropic cooperativity with S0.5 values of 50 +/- 12 microM for cyclic AMP and 35 +/- 15 microM for cyclic GMP and Hill coefficients of 1.2 to 1.5 for both nucleotides. Low levels of cyclic GMP stimulate the rate of cyclic AMP hydrolysis from 3- to 10-fold. The activity of this phosphodiesterase is not stimulated by the calcium binding protein, calmodulin. The cyclic GMP stimulation of cyclic AMP hydrolysis by this phosphodiesterase may provide a possible regulatory link between the metabolism of these two nucleotides in platelets.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1990

Membrane expression of platelet calpain.

Platelet calpain has many platelet substrates, including external membrane proteins. We thus investigated whether platelet calpain II was associated with platelet membranes in unstimulated and thrombin-activated platelets. A monospecific, goat polyclonal antibody was reared to purified platelet calpain II. Sixteen whole platelet lysates were found to contain 4.5 +/- 0.7 micrograms calpain antigen II per 10(8) platelets (mean +/- SEM) as determined by a competitive enzyme-linked immunosorbent assay. Using the dipeptide fluorogenic substrate, Suc-Leu-Tyr-MCA, 17 human platelet lysates contained 3.6 +/- 0.4 micrograms calpain activity per 10(8) platelets. Platelet calpain II was associated with the Triton X-100 insoluble platelet cytoskeletons from both unstimulated and thrombin-activated platelets. When compared with the total cell content of platelet calpain II, calpain antigen (10% to 13%) and calpain activity (24% to 28%) was associated with platelet cytoskeletons in unstimulated and thrombin-activated platelets, respectively. On immunoblot, the heavy chain (80 Kd) of calpain II was detected in platelet cytoskeletons. Subcellular fractionation studies on both unstimulated and thrombin-activated platelets, revealed that half of the total platelet calpain II antigen was associated with cytosol, and the other half was associated with the membrane fraction. Platelet calpain II was not seen on the surface of unstimulated, paraformaldehyde fixed platelets by immunofluorescence. However, on thrombin-activated platelets, rim immunofluorescence was seen, indicating that activated platelets externalize their calpain. This observation was confirmed by the finding that about 2,000 molecules per platelet of an 125I-anti-calpain II Fab' specifically bound to thrombin-activated but not unstimulated platelets. Both dibucaine (1 mmol/L) and platelet activating factor (1.86 mumol/L) in the absence of external Ca++, but not collagen (5 micrograms/mL) or ionophore A23187 (2.5 mumol/L) in the absence of external Ca++, were also able to externalize platelet calpain II antigen, as indicated by a similar level of specific 125I-anti-calpain II Fab'-platelet binding. These combined studies indicate that platelet calpain II is a major protein, comprising 2% of total platelet protein, a substantial portion of which is membrane-associated. When platelets are activated by thrombin and platelet activating factor, calpain II antigen also becomes present on the external platelet surface.

Antibodies↗
PubMed1990

Three new potential cAMP affinity labels. Inactivation of human platelet low Km cAMP phosphodiesterase by 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate.

Three new analogues of cAMP have been synthesized and characterized: 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate (2-BDB-TcAMP), 2-[(3-bromo-2-oxopropyl)thio]-adenosine 3',5'-cyclic monophosphate (2-BOP-tcAMP), and 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate (8-BDB-TcAMP). The bromoketo moiety has the ability to react with the nucleophilic side chains of several amino acids, while the dioxobutyl group can interact with arginine. These cAMP analogues were tested for their ability to inactivate the low Km (high affinity) cAMP phosphodiesterase from human platelets. The 2-BDB-TcAMP and 2-BOP-TcAMP were competitive inhibitors of cAMP hydrolysis by the phosphodiesterase with Ki values of 0.96 +/- 0.12 and 0.70 +/- 0.12 microM, respectively. However, 2-BDB-TcAMP and 2-BOP-TcAMP did not irreversibly inactivate the phosphodiesterase at pH values from 6.0 to 7.5 and at concentrations up to 10 mM. These results indicate that although the 2-substituted TcAMP analogues bind to the enzyme, there are no reactive amino acids in the vicinity of the 2-position of the cAMP binding site. In contrast, incubation of the platelet low Km cAMP phosphodiesterase with 8-BDB-TcAMP resulted in a time-dependent, irreversible inactivation of the enzyme with a second-order rate constant of 0.031 +/- 0.009 min-1 mM1. Addition of the substrates, cAMP and cGMP, and the product, AMP, to the reaction mixture resulted in marked decreases in the inactivation rate, suggesting that the inactivation was due to reaction at the active site of the phosphodiesterase.(ABSTRACT TRUNCATED AT 250 WORDS)

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1990

Human platelet factor V is crosslinked to actin by FXIIIa during platelet activation by thrombin.

Although it has been established that factor V (FV) becomes associated irreversibly with the platelet cytoskeleton after stimulation with thrombin, the chemical nature of this complex is not known. Factor V has recently been demonstrated to be a substrate for factor XIIIa and to form factor V oligomers. We now show that thrombin-activated 125I-FV specifically links to a single protein (43 kDa) of the solubilized platelet membrane in a reaction which requires Ca++ and factor XIIIa. In a purified system, FV, activated by thrombin, forms covalently linked high molecular complexes with 125I-actin catalyzed by factor XIIIa. The site of crosslinking of actin was the factor V fragments, 150 kDa (connecting peptide, C1) and its parent molecule 200 kDa (B). Using radiolabeled actin and unlabeled FV, factor XIIIa catalysed the formation of both homopolymers and heteropolymers. Unlabeled actin was found to compete with radiolabeled FV as a substrate for FXIIIa. To evaluate the biological significance of the crosslinking of factor V to actin, intact platelets were treated with B10 (monoclonal antibody to C1), or monospecific polyclonal antibodies to actin or FXIII. After stimulation with thrombin, the cytoskeleton (material insoluble in Triton X-100) showed markedly decreased 125I-FV in the crosslinked complexes. FV coagulant activity associated with platelet cytoskeleton was also diminished following incubation with an antibody to actin, factor XIII, or B10. These data suggest that FV, through the C1 domain, is crosslinked to actin in the cytoskeleton of thrombin-treated platelets. Activated factor XIII may play a role in plasma FV-platelet interaction as well as the expression of FV derived from the alpha-granules on the cytoskeleton during platelet stimulation.

Actins↗
PubMed1990

Interactions between the contact system, neutrophils and fibrinogen.

Since plasma kallikrein activates human neutrophils, and in plasma prekallikrein (PK) circulates complexed with high molecular weight kininogen (HK), we determined whether HK could mediate kallikrein's association with neutrophils. Human neutrophils were found to possess surface-membrane binding sites for HK but no internalization was detected at 37 degrees C. 125I-HK binding to neutrophils was dependent upon Zn++, specific, saturable and reversible with a Kd of 9-18 nM and 40,000-70,000 sites per cell. Furthermore, HK found in neutrophils (240 ng/10(7) neutrophils) also served as a cofactor for HNE secretion since neutrophils deficient in HK have reduced HNE secretion when stimulated in plasma deficient in HK or with purified kallikrein. Thus, neutrophil surface HK may serve as a receptor for kallikrein. Fibrinogen inhibited 125I-fibrinogen bound specifically and reversibly to human neutrophils. Zn++ (50 microM) was required for binding of 125I-fibrinogen to neutrophils and the addition of Ca++ (2 mM) increased the binding 2-fold. Excess HK completely inhibited binding of and displaced labeled fibrinogen as well as unlabeled fibrinogen. Binding of 125I-fibrinogen was saturable with an apparent Kd of 170 nM and 140,000 sites/neutrophil. The binding of 125I-fibrinogen to neutrophils was not inhibited by the peptide RGDS derived from the alpha-chain of fibrinogen, nor by the monoclonal antibodies (MAB) 10E5 to the platelet glycoprotein IIb/IIIa heterodimer. Fibrinogen binding was inhibited by a gamma-chain peptide CYGHHLGGAKQAGDV and by MAB OKM1 but was not inhibited by OKM10, a MAB to a different domain of the adhesion glycoprotein Mac-1 (CR3). HK binding to neutrophils was not inhibited by OKM1. These observations were consistent with a further finding that fibrinogen is a noncompetitive inhibitor of 125I-HK binding to neutrophils. These studies indicate that fibrinogen specifically binds to an integrin receptor (Mac-1) on the neutrophil surface through the carboxy terminal of the gamma-chain and that HK inhibits this interaction.

Adult↗
PubMed1990

Platelet and neutrophil activation in cardiopulmonary bypass.

My colleagues and I have employed a simulated extracorporeal circuit to help define blood cell changes during clinical cardiopulmonary bypass. Platelet count decreases sharply due to temporary adhesion to the circuit. Platelets degranulate, synthesize and release thromboxane A2, and lose the ability to aggregate with adenosine diphosphate and epinephrine. These changes are also due to the loss of alpha 2-adrenergic and fibrinogen receptors. The neutrophil count decreases to a lesser extent, but neutrophils also are stimulated to secrete lactoferrin and elastase concomitant with activation of plasma kallikrein. Although lidocaine can inhibit the neutrophil activation and prostacyclin can inhibit the platelet stimulation, prostaglandin E1 appears to prevent both neutrophil and platelet alterations.

Cardiopulmonary Bypass↗
PubMed1990

Aggregin: a platelet ADP receptor that mediates activation.

ADP is known to induce platelet shape change, aggregation, and exposure of fibrinogen binding sites as well as inhibit stimulated adenylate cyclase. The platelet is unique in that its purinergic receptor prefers ADP over ATP, which functions as a competitive antagonist. The affinity reagent, 5'-p-fluorosulfonylbenzoyl adenosine (FSBA), has been used to covalently label a single membrane protein, aggregin, on the external platelet surface with mol wt of 100 kDa. Concomitant with incorporation of FSBA, ADP-induced shape change, aggregation, and fibrinogen binding is inhibited. FSBA is also a weak agonist at short times and high concentration, which suggests that prior noncovalent binding to aggregin takes place before covalent modification. Aggregin differs from platelet glycoprotein IIIa in its physical and immunochemical properties. Aggregin is distinct from the receptor coupled to adenylate cyclase. Using FSBA as a probe, platelet aggregation by thromboxane A2 analogs and collagen was shown to be dependent on ADP but not the shape change induced by these agonists. Binding to aggregin is required for epinephrine-induced aggregation. In turn, epinephrine increases the affinity of ADP for its receptor. Thrombin at concentrations greater than 2 nM (0.2 units/ml) stimulates platelet aggregation independent of ADP, but by raising cytoplasmic Ca2+ it activates platelet calpain, which in turn cleaves aggregin. Thus aggregin, in addition to serving as the ADP receptor linked to shape change and aggregation, plays a role in fibrinogen receptor latency that is relieved entirely by ADP binding to or proteolysis of aggregin.

Adenosine Diphosphate↗
PubMed1990

Plasmin-induced platelet aggregation is accompanied by cleavage of aggregin and indirectly mediated by calpain.

We recently reported that thrombin-induced platelet aggregation 1) is accompanied by cleavage of aggregin, a 100-kDa membrane protein and a putative ADP receptor, 2) is indirectly mediated by intracellularly activated calpain, and 3) requires the occupancy of high-affinity thrombin receptors. Because of the similarities between responses after platelet activation induced by thrombin and plasmin (greater than or equal to 1.0 casein unit/ml), we investigated whether or not plasmin-induced platelet aggregation proceeds by the same mechanism that underlies thrombin-induced platelet aggregation. We found that the rate of plasmin-induced aggregation of washed intact platelets and that of platelets modified by 5'-p-fluorosulfonylbenzoyladenosine (FSBA, an affinity analogue of ADP, which covalently modifies aggregin) were similar, indicating that the aggregation is independent of the ADP effect. Plasmin completely cleaved [3H]FSBA-labeled aggregin in intact platelets. A mixture of metabolic inhibitors (2-deoxy-D-glucose, gluconolactone, and antimycin A) completely inhibited plasmin-induced platelet aggregation and plasmin-induced cleavage of aggregin, demonstrating that an energy-requiring step is involved in the reaction. The synthetic hexapeptide affinity reagent Phe-Gln-Val-Val-Cys(NpyS)-Gly-NH2 (NpyS = 3-nitro-2-thiopyridine), a potent and specific inhibitor of thrombin-induced platelet aggregation and platelet calpain, completely inhibited plasmin-induced platelet aggregation and plasmin-induced cleavage of aggregin. These results suggest that, like thrombin, plasmin-induced platelet aggregation is accompanied by the cleavage of aggregin and these responses are indirectly mediated by the intracellularly activated calpain.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Platelet receptors.

This article highlights the increasing knowledge of the biochemistry, pathology, and cell and molecular biology of platelet receptors. A receptor for ADP has been identified using the affinity label FSBA as aggregin, a 100-kd membrane protein, responsible for shape change, aggregation, and exposure of fibrinogen binding sites. A variety of putative receptors for collagen have been described with GP Ia and GP IV receiving the most attention recently. A thromboxane A2 receptor has been identified using receptor antagonists and photoaffinity labels. The alpha 2-adrenergic receptor has been cloned and expressed. The platelet thrombin receptor has been identified as GP Ib. Following binding of thrombin to this receptor, activation of calpain occurs with cleavage of aggregin leading to exposure of GP IIb/IIIa and platelet aggregation. Isolation, expression, or both of the ADP, collagen, and thrombin receptors as single gene products of the human platelet responsible for activation, and more complete understanding of stimulus-response coupling should allow for greater specificity of drugs with selective therapeutic actions.

Adenosine Diphosphate↗
PubMed1989

Cleavage of a 100 kDa membrane protein (aggregin) during thrombin-induced platelet aggregation is mediated by the high affinity thrombin receptors.

Thrombin-induced platelet aggregation is accompanied by cleavage of aggregin, a surface membrane protein (Mr = 100 kDa), and is mediated by the intracellular activation of calpain. We now find that agents that increase intracellular levels of platelet cAMP by stimulating adenylate cyclase, also inhibit thrombin binding and platelet activation by destabilizing thrombin receptors on the platelet surface. Iloprost (a stable analog of PGI2) and forskolin each completely inhibited platelet aggregation by 2 nM thrombin and markedly decreased cleavage of aggregin. Thrombin inactivated by D-phenylalanine-L-prolyl-L-arginine chloromethyl ketone (PPACK-thrombin) binds to the highest affinity site for thrombin on the platelet surface, but thrombin modified by N alpha-tosyl-L-lysine chloromethylketone (TLCK-thrombin) does not. We now demonstrate that preincubation of platelets with PPACK-thrombin blocked platelet aggregation and cleavage of aggregin induced by 2 nM thrombin. In contrast, TLCK-thrombin neither blocked platelet aggregation nor the cleavage of aggregin. These results show that a) platelet aggregation and cleavage of aggregin by thrombin (2nm) involves the occupancy of high affinity alpha-thrombin receptors on the platelet surface, and b) stimulators of adenylate cyclase which increase cAMP, inhibit thrombin-induced platelet aggregation and cleavage of aggregin by mechanisms which include inhibiting the binding of thrombin to its receptors.

Calpain↗
PubMed1989

Mapping of a putative surface-binding site of human coagulation factor XII.

We have localized the binding epitope(s) of two murine monoclonal antibodies (B7C9 and P5-2-1) that were shown previously to inhibit the activation of human coagulation factor XII by negatively charged surfaces. A factor XII cDNA expression library in lambda gt11 was screened with antibody B7C9, and 16 immunoreactive bacteriophage were isolated. Fusion proteins from each of the recombinant phage were reactive with both monoclonal antibodies. Two of the phage cDNA inserts were found to code for amino acid residues -6-+31 and +1-+47 of factor XII, respectively, thereby defining the limits of the antigenic peptide to amino acids +1-+31. Each of the remaining 14 recombinant phage contained longer factor XII cDNA inserts that included sequences coding for the amino-terminal 31 amino acid residues. These results were confirmed by direct binding of antibody B7C9 to synthetic peptides containing amino acids 1-14 and 1-28 of factor XII. Further experiments with a set of nested peptides also indicated that amino acid residues 1-4 were essential but not sufficient for binding of B7C9 to the peptides. Hydrophobicity analysis of the amino-terminal region of plasma factor XII revealed a highly hydrophilic region between amino acid residues 5 and 15 that contained positively charged lysine residues at positions 8, 11, and 13. We conclude that a major epitope(s) recognized by monoclonal antibodies B7C9 and P5-2-1 is present in the amino-terminal 28 amino acids of factor XII. It is proposed that binding of these antibodies to factor XII blocks interaction of the positively charged region between residues 5 and 15 with negatively charged surfaces, thereby inhibiting activation.

Antibodies, Monoclonal↗