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R W Colman

Publications and source records attributed to R W Colman.

At least 127 records · Page 7Linked to original sources
PubMed1994

Thrombin-stimulated human platelets express molecular forms of alpha-granule factor V (FV), which differ from FVa.

Binding of 125I-Fab fragments of chain-specific antibodies indicate that both heavy and light chains of alpha-granule factor Va (FVa) were externalized on the platelet membrane after stimulation with thrombin. Using a Mab against the activation peptide of factor V (FV), the epitope appears on the stimulated platelet surface. Half as much light chain and heavy chain (FVa) was expressed compared to the activation peptide, suggesting that expression of alpha-granule FV occurs after thrombin stimulation. Using an ELISA, we find that 32% of alpha-granule FV was released and 68% is retained in the platelet pellet. Immunoblots of platelets indicate that FV exists in 200 kDa und 150 kDa forms, representing incomplete cleavage, while the releasate demonstrates a more complete cleavage by proteases. We conclude that expression of alpha-granule FV is quantitatively greater than that released and exists in molecular forms which cannot be completely explained by the binding of FVa.

Amino Acid Sequence↗
PubMed1994

Structure-function correlates of human high molecular weight kininogen.

1. The first documented mutation responsible for total human kininogen deficiency has been characterized as a single base change from CGA to TGA in exon 5, resulting in an Arg-->Stop mutation inherited as an autosomal recessive trait. 2. The surface binding domain (D5) of high molecular weight kininogen (HK) was mapped by deletion mutagenesis into two regions, the first rich in histidine and glycine, and the second in histidine, glycine and lysine. 3. The structural requirements of domain 2 to inhibit calpain have been mapped using affinity labels and conformationally constrained peptides. The surface loops consist of a binding region QVVAG and an inhibitory region C-terminal consisting of a sequence containing the sequence NAYI. 4. Domain 3 contains structural components capable of inhibiting the binding of thrombin to platelets. 5. Each domain of HK has a unique function which contributes to different aspects of the inflammatory reactions mediated by plasma protein and blood cells.

Amino Acid Sequence↗
PubMed1994

Platelet thrombospondin interactions with human high and low molecular weight kininogens.

Multifunctional proteins, e.g. high molecular weight kininogen (HK, 120 kDa) and the homotrimer, thrombospondin (TSP, 540 kDa), which have more than one domain on a single polypeptide chain, are particularly well-suited to be structural elements of extracellular matrices because of their ability to bind to several macromolecules. We now demonstrate that 125I-high molecular weight kininogen (HKa) cleaved by purified kallikrein forms a complex with purified intact platelet TSP (540 kDa). HK also complexed with a proteolytic fragment (450 kDa) of TSP, lacking its three identical heparin-binding domains (HBD, 30 kDa), but failed to bind to a more extensively proteolysed molecule (210 kDa) lacking the C-terminal globular domain indicating that the binding on TSP-450 kDa is confined to the C-terminus. The binding of HK to intact TSP and to its 450 kDa fragment was of high affinity (Kd = 17-52 nM), specific, concentration dependent and saturable. Furthermore, we found both forms of the light chain (LC) of HK (56 and 46 kDa) resulting from cleavage by plasma kallikrein bound to both intact TSP and HBD independent of the presence of calcium ions. However, neither the epitope recognized by monoclonal antibody (MAb) C11C1 on domain 5 nor the prekallikrein binding site on domain 6 are involved, suggesting that the intervening proline-rich region may be the site of interaction. The heavy chain (HC) of HK required ionized calcium to bind to intact TSP or its 450 kDa homotrimer fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Platelets↗
PubMed1993

Contiguous binding and inhibitory sites on kininogens required for the inhibition of platelet calpain.

Both high molecular weight kininogen (HK) and low molecular weight kininogens (LK) are potent tight binding inhibitors of platelet calpain (Ki = 2 nM), but the molecular basis for the inhibitory function is not well delineated. The amino acid sequences of the calpain inhibitory domain 2 from human and rat HK were compared for homology with the noninhibitory domains from human and rat domain 3 and from domain 2 of rat T-kininogen, and two areas of nonconserved differences were detected. Computer three-dimensional models were constructed on a template built using the x-ray crystallographic data for cystatin, an evolutionary precursor of HK. Two nonconserved regions in the calpain inhibitory domains flank the highly conserved motif QVVAG to form a continuous surface for interaction with cysteine proteases. Three peptide sequences, components of the modeled surface, were chosen for synthesis from HK D-2: VHPISTQSPDLE (peptide 146-156, NH2-terminal), CTDNAYIDIQLRIASFSQNC (peptide 229-248, COOH-terminal), and CQRQVVAGLNFRIC (185-189, central) containing QVVAG. This last peptide differs from the natural sequence by substitutions of A185C and T195C. Peptides 185-198 and 229-248 were folded by air oxidation of their cysteine residues and then tested for their ability to inhibit calpain and papain. The folded peptide 229-248 inhibited calpain with an IC50 35 microM and unfolding reduced this effect. The folded peptide 185-198 did not inhibit calpain, but when preincubated with calpain, could block the inhibition by HK indicating a probable enzyme binding site. Peptide 146-157 did not inhibit calpain but could inhibit papain with an IC50 of 20 microM. We have thus defined separate binding and inhibitory sequences on HK which form a contiguous surface for thiol protease interactions.

Amino Acid Sequence↗
PubMed1993

Genetic basis of total kininogen deficiency in Williams' trait.

High and low molecular weight kininogens (HK and LK) modulate inflammatory responses, serve as precursors of kinins, inhibit cysteine proteinases, and modulate thrombin activation of platelets. Differential splicing yields two different mRNAs for HK and LK. We report the first molecular characterization of a kininogen gene mutation in a patient lacking LK and HK. No gross DNA deletion or insertion was detected by Southern blots, and LK and HK mRNAs were normal size on Northern blots. Exon sequences amplified by polymerase chain reaction showed a C-->T transition at nucleotide 587, resulting in a CGA (Arg)--> TGA (Stop) mutation in exon 5 before the splice site in exon 10, thus preventing synthesis of both HK and LK. The mutation eliminated the recognition site of the restriction enzyme Csp45I. Results of Csp45I digestion of the polymerase chain reaction-amplified exon 5 DNA fragment of the patient (lacking kininogens), three daughters (approximately 50% HK), and 1 granddaughter (normal HK) revealed that the patient was homozygous, while the three daughters were heterozygous, and the granddaughter was normal. We conclude that a single base mutation in the kininogen gene exon 5 was responsible for kininogen deficiency in the Williams family.

Base Sequence↗
PubMed1993

Specificity of the sequence in Phe-Gln-Val-Val-Cys (-3-nitro-2-pyridinesulfenyl)-Gly-NH2--a selective inhibitor of thrombin-induced platelet aggregation.

Thrombin-induced platelet aggregation is mediated in part by the intracellularly activated calpain expressed onto the external side of the membrane. We have previously shown that P1, Phe-Gln-Val-Val-Cys(Npys)-Gly-NH2 [Npys = 3-nitro-2-pyridinesulfenyl], an affinity analog corresponding to the highly conserved sequence Gln-Val-Val-Ala-Gly-NH2, present in domains 2 and 3 of human kininogens, was an irreversible inhibitor of platelet calpain (second-order rate constant = 5.85 mM-1 s-1). P1 also selectively blocked thrombin-induced platelet aggregation. We have now synthesized twenty-three other peptides, analogous to P1, and evaluated them to define the specificity of the amino acid sequence in P1 to selectively block thrombin-induced platelet aggregation. We find that replacement by Leu of Val and by Tyr of Phe adjacent to Gln is minimally tolerated and the resulting peptides are partially effective in selectively blocking thrombin-induced platelet aggregation. The presence of valine adjacent to cysteine in P1 is essential for the inhibitor to selectively block thrombin-induced platelet aggregation. The presence of valine adjacent to cysteine in P1 is essential for the inhibitor to selectively block thrombin-induced platelet aggregation. Extensions of the N-terminal sequence in P1 did not improve its selectivity. Ac-Ala-Gln-Val-Val-Ala-Gly-NH2 (Ac, acetyl), a peptide containing the conserved sequence but lacking the Npys function, neither inhibited platelet calpain nor platelet aggregation induced by thrombin. Presence of the peptide sequence and Npys function are both required in P1 for its selective action in inhibiting platelet aggregation induced by thrombin.

Amino Acid Sequence↗
PubMed1993

Characterization of monocyte-associated factor V.

We demonstrate that in addition to possessing binding sites for intact factor V (FV), unstimulated peripheral blood monocytes also express activated factor V (FVa) on their surfaces. FVa was identified on the monocyte surface by monoclonal antibody B38 recognizing FVa light chain and by human oligoclonal antibodies H1 (to FVa light chain) and H2 (to FVa heavy chain) using immunofluorescence microscopy and flow cytometry. On Western blots, partially cleaved FV could be identified as a 220 kDa band in lysates of monocytes. In addition to surface expression of FVa, monocytes also contain intracellular FV as detected only after permeabilization by Triton X-100 by monoclonal antibody B10 directed specifically to the Cl domain not present in FVa. We sought to determine whether the presence of FV in peripheral blood monocytes is a result of de novo synthesis. Using in situ hybridization, no FV mRNA could be detected in monocytes, while in parallel control studies, factor V mRNA was detectable in Hep G2 cells and CD18 mRNA in monocytes. In addition, using reverse transcriptase and the polymerase chain reaction, no FV mRNA was detected in mononuclear cells or in U937 cells, but mRNA for factor V was present in Hep G2 cells using the same techniques. These data suggest that FV is present in human monocytes, presumably acquired by binding of plasma FV, and that the presence of this critical coagulation factor is not due to de novo synthesis.

Antibodies, Monoclonal↗
PubMed1993

Activation of the kallikrein-kinin system after endotoxin administration to normal human volunteers.

The objective of this study was to determine the role of the kallikrein-kinin system in healthy humans after intravenous administration of either Escherichia coli endotoxin or saline. We studied a total of 18 healthy nonsmoking volunteers, 23 to 38 years old, in an open-label study at the Critical Care Medicine Department, Clinical Center, National Institutes of Health (Bethesda, MD) in which some of the patients served as their own controls. After baseline data collection, the subjects received intravenously either E coli endotoxin (n = 15, 4 ng/kg of body weight) or saline (n = 8, controls). Signs, symptoms, systemic blood pressure, factor XII, plasma prekallikrein (PK), factor XI (FXI), antithrombin III (AT-III), high molecular weight kininogen (HK), and alpha 2-macroglobulin-kallikrein complexes were measured at baseline and 1, 2, 3, 5, and 24 hours after injection of either saline or endotoxin. After infusion of endotoxin, we found the functional plasma levels of FXI decreased at 2 hours (P < .05) and at 5 hours (P < .05). Functional PK was significantly depressed by 2 hours (P < .05), at 5 hours (P < .05), and at 24 hours (P < .01), whereas the PK antigen was only low at 5 hours (P < .05). These changes were accompanied by a significant increase in circulating alpha 2-macroglobulin-kallikrein complexes at 3 hours (P < .05) and 5 hours (P < .01). No significant changes occurred in the plasma levels of factor XII or HK. We concluded that clinical response to intravenous endotoxin in healthy human volunteers is associated with activation of the kallikrein-kinin systems. Further investigation is needed with specific inhibitors of the kallikrein-kinin system to define its primary or secondary role in the endotoxin-mediated reactions.

Adult↗
PubMed1993

Modulation of thrombin-induced platelet aggregation by inhibition of calpain by a synthetic peptide derived from the thiol-protease inhibitory sequence of kininogens and S-(3-nitro-2-pyridinesulfenyl)-cysteine.

Thrombin-induced platelet aggregation has been suggested to play an important role in reocclusion following thrombolytic therapy of angioplasty for treatment of myocardial infarction. We previously demonstrated that aggregation of washed platelets by thrombin is accompanied by cleavage of aggregin, a putative ADP receptor, and that these events are indirectly mediated by calpain, expressed on the surface of the external membrane. High-molecular-mass kininogen (HK) contains, in its heavy chain, domain 2, which is responsible for its action as a potent inhibitor of platelet calpain. Domain 3 of the heavy chain of HK directly inhibits binding of thrombin to platelets, confounding mechanistic studies using the entire molecule. Moreover, HK, a protease of 120 kDa, is unsuitable as a potential pharmacological agent. The highly conserved sequence Gln-Val-Val-Ala-Gly, present in HK and its evolutionary precursors, the cystatins, is thought to be involved in the binding of cysteine proteases but is, itself, not inhibitory. An affinity analog, Phe-Gln-Val-Val-Cys(Npys)-Gly-NH2(Npys, 3-nitro-2-sulfenylpyridine), P1, corresponding to the thiol-protease-binding sequence in HK and containing a ligand, Npys, that can react with the free sulfhydryl group in the active site of calpain, was synthesized. P1 was an irreversible inhibitor of platelet calpain. P1 selectively inhibited thrombin-induced aggregation of washed platelets and platelets in plasma, but did not inhibit the aggregatory effects of other platelet agonists. P1 did not inhibit the amidolytic activity and coagulant activity of thrombin. Unlike HK, P1 did not inhibit binding of thrombin to washed platelets. P1 did not inhibit thrombin-induced platelet-shape change. P1 neither raised intracellular levels of cAMP nor did it interfere with the ability of thrombin to antagonize the rise in intracellular levels of cAMP induced by iloprost, an analog of prostaglandin I2. The design and synthesis of P1 could leave to the development of a new class of inhibitors that selectively block thrombin-induced platelet aggregation while sparing other functions of this pathophysiological protease and without inhibiting the action of other platelet agonists.

Amino Acid Sequence↗
PubMed1993

Purification and characterization of a potent 70-kDa thiol lysyl-proteinase (Lys-gingivain) from Porphyromonas gingivalis that cleaves kininogens and fibrinogen.

We isolated an enzyme from a major periodontal pathogen, Porphyromonas gingivalis (also called Bacteroides gingivalis), that is capable of initially increasing the coagulant activity of high molecular weight kininogen (HK), releasing bradykinin from HK and low molecular weight kininogen (LK), and destroying the light chain (coagulant portion) of HK. This enzyme, a membrane-bound thiol proteinase that preferentially cleaves the P1-Lys position of tripeptide substrates, is also able to rapidly render fibrinogen nonclottable. We will refer to this enzyme as lys-gingivain because of its origin from P. gingivalis, its classification as a thiol proteinase, and its action as a lysyl-amidase. The activity of lys-gingivain is enhanced by beta-mercaptoethanol, and the enzyme has a molecular mass of 68-70 kDa, a pH optimum of 7.4, and is not inactivated by plasma protease inhibitors. The second-order rate constant for the destruction of the coagulant activity of the HK light chain (surface-binding domain) at 23 degrees C is 2.3 x 10(7) M-1 s-1, and, for cleavages that render fibrinogen unclottable, is 2.05 x 10(6) M-1 s-1. These data suggest that lys-gingivain is a very potent proteinase that would be fully functional in anaerobic periodontal crevices and might participate in the pathogenesis of periodontitis. Lys-gingivain appears to be the most potent kininogenase and fibrase to be described to date.

Amino Acid Sequence↗
PubMed1993

Deletion mutagenesis of high molecular weight kininogen light chain. Identification of two anionic surface binding subdomains.

The light chain (LC) of cleaved high molecular weight kininogen (HK) binds to anionic surfaces as well as the zymogens prekallikrein and factor XI and thus accelerates activation of the kallikrein-kinin, fibrinolytic, and coagulation pathways. The binding sites on HK LC for factor XI (amino acid residues 574-631) and prekallikrein (residues 583-613) have been localized to domain 6. Domain 5 (residues 438-520) has been postulated to contain the anionic surface binding subdomain. In order to define this subdomain we have expressed HK LC (residues Lys438-Ser644) as a fusion protein with glutathione-S-transferase (GST) in Escherichia coli and generated various HK LC deletion mutants. The recombinant HK LC (rHK LC) and various HK LC fragments were purified as GST fusion proteins by glutathione-Sepharose affinity chromatography from bacterial cell extracts. The rHK LC and recombinant fragments His459-Ser644, Glu466-Ser644, Leu483-Ser644, His493-Ser644, Lys438-Asp492, Lys438-Ser531, and His493-Lys520 inhibited 125I-HKa binding to kaolin, a model anionic surface used in the contact system, in a concentration-dependent manner. Deletion mutant proteins lacking domain 5, Thr521-Ser644 and Ser583-Ser644, did not inhibit the radiolabeled HKa binding to kaolin. The rHK LC and recombinant fragments Lys438-Asp492, Lys438-Ser531, His493-Ser644, His493-Lys520, Thr521-Ser644, and Ser583-Ser644 were radiolabeled with 125I and were then tested for their ability to bind to kaolin in the presence of fibrinogen and albumin. Except for the Thr521-Ser644 and Ser583-Ser644 fragments, all other radiolabeled HK LC deletion mutant proteins and rHK LC bound to kaolin in a concentration-dependent manner. This binding to kaolin was specific since it was inhibited by the addition of excess unlabeled HKa. The rHK LC, His493-Ser644 and delta 493-520 HK LC have coagulant activity, while other deletion mutant proteins did not exhibit coagulant properties. We conclude that there are at least two anionic surface binding subdomains, one in the histidine-glycine-rich region (Lys438-Asp492) and the other in the histidine-glycine-lysine-rich region (His493-Lys520), in the domain 5 of HK LC. Either subdomain, in the presence of the zymogen binding domain 6, is sufficient to impart coagulant activity to HK LC, while the presence of both did not increase the coagulant activity of HK LC additively.

Amino Acid Sequence↗