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Biomedical subjects

R W Colman

Publications and source records attributed to R W Colman.

At least 235 records · Page 13Linked to original sources
PubMed1987

A monoclonal antibody recognizing an icosapeptide sequence in the heavy chain of human factor XII inhibits surface-catalyzed activation.

A monoclonal antibody (mAb B7C9) to human factor XII was raised in murine somatic cell using purified factor XII antigen. The purified antibody was subtyped IgG1 kappa and had a KD of 9.8 nM for antigen factor XII. Functional studies indicated that mAb B7C9 blocks surface-mediated coagulant activity of factor XII but not the amidolytic activity of factor XIIa against the small substrate H-D-Pro-Phe-Arg-p-nitroanilide (S-2302), suggesting that the mAb B7C9 epitope is located at or near the surface binding domain of the heavy chain region of factor XII. Western blot analysis indicated that the antibody reacts with factor XII and the heavy chain of factor XIIa. Affinity isolation of factor XII peptides, produced after cleavage by kallikrein, resulted in three factor XII heavy chain domain segments that were identified in the known factor XII sequence by limited N-terminal analysis. The epitope was located to a 20-amino acid sequence of 2.5 kDa in the heavy chain of factor XII which is the putative surface binding region of factor XII. The 2.5-kDa peptide was synthesized and demonstrated to react with mAb B7C9. mAb B7C9 was immobilized on an affinity resin and was successfully utilized to purify functionally active factor XII from plasma.

Animals↗
PubMed1987

Determination of the bifunctional properties of high molecular weight kininogen by studies with monoclonal antibodies directed to each of its chains.

In normal human plasma two forms of kininogen exist, low molecular weight kininogen (LMWK) and high molecular weight kininogen (HMWK). When these proteins are cleaved they are found to have a common heavy chain and bradykinin, but each has a unique light chain. Monoclonal antibodies to the heavy and light chains of HMWK have been developed, and the effects of each on the function of this protein are defined. Initial studies showed that an antibody, C11C1, completely neutralized the coagulant activity of plasma HMWK whereas another antibody, 2B5, did not. On a competitive enzyme-linked immunosorbent assay (CELISA) the C11C1 antibody was consumed by kininogen antigen in normal plasma but not by kininogen antigen in HMWK-deficient plasma. On immunoblot, the C11C1 antibody recognized one kininogen protein in normal plasma and did not recognize any kininogen antigen in HMWK-deficient plasma. These combined studies indicated that the C11C1 antibody was directed to an epitope on the unique 46-kDa light chain of HMWK. In contrast, the 2B5 antibody on a CELISA was consumed by kininogen antigen in both normal plasma and HMWK-deficient plasma but not by total kininogen-deficient plasma. On immunoblot, the 2B5 antibody recognized both kininogens in normal plasma but only LMWK in HMWK-deficient plasma. These combined studies indicated that the 2B5 antibody was directed to the common 64-kDa heavy chain of the plasma kininogens. Utilizing direct binding studies or competition kinetic experiments, the 2B5 and C11C1 antibodies bound with high affinity (1.71 and 0.77 nM, respectively) to their antigenic determinants on the HMWK molecule. The 2B5 antibody did neutralize the ability of HMWK to inhibit platelet calpain. These studies with monoclonal antibodies directed to each of the HMWK chains indicate that HMWK is a bifunctional molecule that can serve as a cofactor for serine zymogen activation and an inhibitor of cysteine proteases.

Animals↗
PubMed1987

Fibrinogen receptors in platelet adhesion to surfaces of extracorporeal circuit.

The role of platelet fibrinogen receptors and platelet-protein interaction in platelet consumption during simulated cardiopulmonary bypass was investigated. In five recirculation experiments, with whole blood, the platelet count fell to 13% of initial values and in two experiments, with blood from patients with Bernard-Soulier syndrome, to 3% of normal values. However, in three experiments with blood from the patients with Glanzmann's thrombasthenia, the platelet count decreased to 69% of initial values. The extent of platelet consumption in normal blood was diminished to only 72% by addition of 0.5 microM prostaglandin E1 (PGE1) (5 experiments) and to 80% by precoating surfaces of the circuit with 2.5% human albumin (5 experiments). beta-Thromboglobulin antigen (beta TG) loss from platelets was associated with thrombocytopenia. The extent of beta TG loss was significantly reduced by the addition of PGE1 to blood or precoating surfaces with albumin. Proteins adsorbed on the surface of the circuit exposed to normal blood were removed with 0.5% Triton X-100. Some of these proteins were identified to be glycoprotein IIIa (GPIIIa) (3.4-4.3 micrograms/ml), beta TG (1.0-1.6 micrograms/ml), and fibrinogen (1.9-3.7 micrograms/ml). The amount of GPIIIa recovered in the Triton X-100 eluates correlated with the number of platelets lost during recirculation. These studies indicate that exposure of fibrinogen receptors associated with GPIIb-GPIIIa complex contributes to platelet consumption during cardiopulmonary bypass.

Blood Platelets↗
PubMed1987

Kinetics of inhibition of human plasma kallikrein by a site-specific modified inhibitor Arg15-aprotinin: evaluation using a microplate system and comparison with other proteases.

Human plasma kallikrein, a product of contact-activated plasma proteolysis, is moderately inhibited by aprotinin, a small polypeptide from bovine lung that has been used as an experimental drug in human disease states. Aprotinin has a Lys residue in the P1 (reactive center) position occupying residue 15. Since kallikrein is an arginine-directed serine protease, we hypothesized that an altered form of aprotinin, Arg15-aprotinin, might be a better inhibitor. Kinetic evaluations were performed in 96-well microplates. We found that the KL (loose or Michaelis-Menten complex) was unchanged by the modification. However, the association rate constant was increased from 1.14 X 10(4) (mol/L)-1s-1 to 1.5 X 10(5) (mol/L)-1s1, thus indicating that the inhibition rate was increased 14-fold for the modified protein. The Ki (at equilibrium) was decreased from 3.2 X 10(-7) mol/L to 1.5 X 10(-8) mol/L after substituting Arg for Lys in the P1 position. Therefore, the modified inhibitor binds to plasma kallikrein more tightly than the natural protein. We also investigated the effect of Arg15-aprotinin on tissue kallikrein, plasmin, factor XIIa, factor XIa, and thrombin and found that the Ki slightly decreased from 5.1 X 10(-7) mol/L to 1.2 X 10(-7) mol/L for tissue kallikrein and slightly decreased from 2 X 10(-8) mol/L to 1 X 10(-8) mol/L for plasmin. Arg15-aprotinin did not inhibit thrombin or factor XIIa, even though both enzymes are arginine-directed serine proteases. However, factor XIa, although it was not inhibited by aprotinin, had a Ki of 3.4 X 10(-8) mol/L for Arg15-aprotinin. Therefore, Arg15-aprotinin is a more effective inhibitor of plasma kallikrein as well as factor XIa but shows minimal preference for plasmin and tissue kallikrein. This study also indicates that it is possible and practical to perform kinetic analyses directly in microplates.

Aprotinin↗
PubMed1987

Epitope mapping of functional domains of human factor Va with human and murine monoclonal antibodies. Evidence for the interaction of heavy chain with factor Xa and calcium.

We have purified a unique neutralizing IgG1, kappa monoclonal antibody (MAb) against factor V (F-V) from a patient's plasma. This MAb (H2) demonstrated specificity for human F-V heavy chain (D), mol wt 105,000. Using an enzyme-linked immunosorbent assay (ELISA) we assessed the competitive binding to F-Va of H2, H1 (human MAb directed to light chain, F1F2), and two murine MAbs, B38 (to F1F2) and B10 (to activation peptide C1). All four antibodies are of high affinity with KD varying from 0.17 to 1.17 X 10(-10) mol/L. They recognized distinct epitopes in F-V. F-Xa competed in a concentration-dependent fashion for binding of H1, H2, and B38 but not B10 to F-V/Va in the absence of phospholipids or platelets. Thus both F1F2 and D polypeptides of F-Va but not C1 interacted with F-Xa. All MAbs bound to F-V/Va in the absence of Ca++. However, free Ca++ (0.1 to 4.0 mmol/L) increased the amount of H1 and H2 bound to factor V/Va, 1.65-fold and 3.65-fold, respectively but had little effect on the binding of either murine MAbs. Prothrombin (20 micrograms/mL to 400 micrograms/mL) in the absence of phospholipid did not inhibit the binding of MAbs. These studies provide evidence for the first time for a direct interaction between human F-Va heavy chain and F-Xa and Ca++ and for the direct binding of F-Xa to F-Va in the absence of phospholipids or platelets and enhance our understanding of functional F-V domains.

Alkaline Phosphatase↗
PubMed1987

A monoclonal anti-human plasma prekallikrein antibody that inhibits activation of prekallikrein by factor XIIa on a surface.

Of five IgGI/k murine monoclonal anti-human prekallikrein antibodies produced (MAbs), MAb 13G11 was selected for studying interaction of prekallikrein with factor XII and high-mol-wt kininogen (HMWK) during activation on a surface. Immunoblots from sodium dodecyl sulfate (SDS) gels showed that this MAb recognizes two variants (88 kd and 85 kd) of prekallikrein and kallikrein both in purified proteins and normal plasma. Under reducing conditions, kallikrein exhibits the epitope on the heavy chain but not on the light chains. Preincubation of MAb 13G11 with prekallikrein (added to prekallikrein-deficient plasma) or with normal plasma inhibited surface activation of prekallikrein 60% to 80%, as judged by amidolytic and coagulant assays. In normal plasma, inhibition by the Fab fragments was 87% of that with the entire MAb. Inhibition was not by competition between the MAb and HMWK, since neither binding of 13G11 to prekallikrein (coated on microtiter plates) was inhibited by an excess of HMWK, nor was hydrolysis of HMWK by kallikrein inhibited by 13G11. Using purified proteins in a system mimicking contact activation, inhibition by 13G11 of prekallikrein activation by factor XIIa, HMWK, and kaolin present was approximately 80%. Decreased inhibition (55% to 25%) occurred without HMWK or when kallikrein was used instead of prekallikrein. Kallikrein activity was not inhibited by 13G11 Fab fragments. These results indicate that the effect of 13G11 in plasma was neither dissociation of prekallikrein-HMWK complex nor a direct effect on kallikrein activity. Similar to the results in plasma, activation of prekallikrein, HMWK present, by factor XIIa bound to kaolin, was inhibited approximately 70% by 13G11. The results suggest a previously unrecognized site on the prekallikrein (heavy chain) required for its interaction with factor XIIa, either shared with the 13G11 epitope or located in very close proximity. The inhibition of kallikrein by intact 13G11 indicates that its binding site on the heavy chain is sterically related to the active site (light chain).

Antibodies, Monoclonal↗
PubMed1987

Direct evidence for the interaction of the nucleotide affinity analog 5'-p-fluorosulfonylbenzoyl adenosine with a platelet ADP receptor.

Previous reports have indicated that the nucleotide affinity analog 5'-p-fluorosulfonylbenzoyl adenosine (FSBA) at concentrations between 40 and 100 mumol/L and at times greater than 20 minutes covalently modifies a single protein component on the external platelet membrane surface and that adenosine diphosphate (ADP) protects against this reaction. That this protein is an ADP receptor linked to platelet activation is shown by FSBA inhibition of ADP-mediated platelet shape change, aggregation, and fibrinogen receptor exposure. In this report, further evidence for the interaction of FSBA with the ADP receptor on platelets is provided by the observation that FSBA at high concentrations (100 to 500 mumol/L) behaves as a weak agonist to produce platelet shape change within one minute as detected by spectroscopic assay and scanning electron microscopy with concomitant phosphorylation of the light chain of platelet myosin. The specificity of FSBA as an agonist is demonstrated by inhibition of FSBA-induced shape change by ATP and the covalent incorporation of SBA as well as the failure of 5'-fluorosulfonylbenozoyl guanosine (FSBG) to cause shape change. In contrast, incubation of platelets with low concentrations of [3H]-FSBA (40 mol/L) is not associated with stimulation of platelet shape change or myosin light chain phosphorylation.

Adenosine↗
PubMed1987

Inhibition of thrombin-induced platelet aggregation by high molecular weight kininogen.

1. Thrombin-induced platelet aggregation and cleavage of [3H]FSBA-labeled aggregin is mediated by platelet calpain. 2. Plasma HMWK inhibits platelet aggregation and cleavage of aggregin in intact platelets, indirectly by thrombin and directly by calpain. 3. Inhibition of thrombin-induced platelet aggregation by HMWK is specific for this proteolytic enzyme. 4. HMWK is the major inhibitor of thrombin-induced activation of platelets in plasma.

Fibrinogen↗
PubMed1987

Evaluation of a microassay for human plasma prekallikrein.

Current methods for determining plasma prekallikrein, one of three zymogens of the contact phase of plasma proteolysis, are laborious and impractical for general use in a clinical laboratory. Therefore, we have developed a simple, reliable assay using commercially available reagents. By use of the substrate H-D-Pro-Phe-Arg-p-nitroanilide-HCI (S-2302), a functional assay, performed in a 96-well microplate, was designed to measure prekallikrein in plasma. Measures were taken to destroy the naturally occurring plasma protease inhibitors of kallikrein without affecting the integrity of the plasma prekallikrein, which allowed complete activation of the zymogen to virtually 100% of predicted activity when compared with that of purified kallikrein. Besides permitting full activation, the use of low pH to destroy critical plasma protease inhibitors allowed the conversion of prekallikrein to kallikrein in as many as 44 plasma samples at one time without the tedious individual timing step usually required to activate each sample. An excellent correlation was found (r = 0.92) when this functional microassay was compared with a functional spectrophotometric assay performed in three subject populations: normal individuals, women receiving oral contraceptives (who frequently exhibit high plasma prekallikrein concentrations), and patients with liver disease (who manifest low plasma prekallikrein levels). This plasma prekallikrein microassay should facilitate the increased determination of plasma prekallikrein in pathophysiologic conditions as well as the monitoring of the progression of various diseases in which contact activation occurs.

Evaluation Studies as Topic↗
PubMed1986

Recent evolutionary divergence of plasma prekallikrein and factor XI.

The evolution in mammals of the zymogens of the contact activation system of coagulation (factor XII, prekallikrein and factor XI) has been investigated. The NH2-terminal sequences of human plasma prekallikrein and the heavy and light chains of kallikrein have been determined and compared with those of bovine prekallikrein and of human and bovine factors XII and XI. The human and bovine NH2-terminal sequences of the light chains (catalytic polypeptide) show striking similarities both among themselves and with those of the catalytic polypeptide chains of other coagulation and digestive proteases, indicating a common origin. Comparison of the NH2-terminal sequences of human prekallikrein with those of the bovine prekallikrein and human bovine factors XIa and XIIa indicates a common origin of the heavy chain of kallikrein and factor XIa, different from that of either factor XIIa or other known amino acid sequences. Ancestral sequences for human and bovine prekallikrein and factor XI, deduced by genetic analysis of the minimum number of base changes indicate that the NH2-terminus of prekallikrein and factor XI have evolved at about the same rate. The estimated time for the gene duplication was about 124 million years ago, a value consistent with the age of the mammals.

Amino Acid Sequence↗
PubMed1986

Platelet ADP receptor and alpha 2-adrenoreceptor interaction. Evidence for an ADP requirement for epinephrine-induced platelet activation and an influence of epinephrine on ADP binding.

The nucleotide affinity analog 5'-p-fluorosulfonylbenzoyl adenosine (FSBA) is a potent irreversible inhibitor of ADP-mediated platelet activation. Utilizing this compound, the role of ADP in epinephrine-mediated platelet activation was evaluated. Pretreatment of platelets with FSBA under conditions producing covalent incorporation was able to completely block epinephrine-stimulated aggregation of human platelets. In addition, the exposure of latent fibrinogen-binding sites by epinephrine was also inhibited in platelets modified by FSBA. The inhibition of epinephrine-mediated activation of the cells was time dependent, reflecting the need for covalent modification of the ADP receptor by FSBA. The inhibitory effect of FSBA was not due to effects on the affinity of binding methyl [3H]yohimbine or the number of platelet alpha 2-adrenergic receptors. Studies of the effect of epinephrine on the ability of ADP to protect against FSBA incorporation demonstrated that epinephrine can increase the affinity of ADP for its receptor 10-fold without affecting the total amount of FSBA covalently bound. This effect of epinephrine is mediated through the alpha 2-adrenoreceptor since the effect can be reversed by the competitive antagonist, methyl yohimbine. These results suggest that promotion of platelet aggregation and the exposure of fibrinogen receptors by epinephrine is dependent on ADP. The mechanism by which epinephrine renders low concentrations of ADP effective appears to be mediated by an increased avidity of the ADP receptor for the nucleotide.

Adenosine↗
PubMed1986

Purification of human factor XII from plasma using zinc chelate affinity chromatography.

Human factor XII (Hageman Factor) was isolated from human plasma to apparent homogeneity using a four step procedure with yields up to 30%. The method, which is more rapid than the current conventional procedures, consists of a 25-50% ammonium sulfate fractionation, two affinity chromatography steps using Zinc Chelate Sepharose, followed by gel filtration. The isolated zymogen factor XII was a single protein component when examined by SDS PAGE with a Mr of 80,000. Activation of zymogen factor XII to its active enzymatic forms by kallikrein resulted in factor XIIa and factor XIIf as observed with factor XII purified by other procedures.

Ammonium Sulfate↗
PubMed1986

Human factor XIa cleaves fibrinogen: effects on structure and function.

Factor XIa, the enzymatic form of the factor XI zymogen, is generated as a result of factor XII-dependent surface activation in plasma. Factor XIa degrades high molecular weight kininogen, its cofactor for activation (which binds factor XIa to the surface), as well as cleaves and activates coagulation factor IX. In this report, we present evidence that factor XIa can also cleave fibrinogen and decrease the thrombin-catalyzed formation of the fibrin clot. Furthermore, the products of factor XIa-digested fibrinogen markedly inhibited the rate of polymerization of fibrin monomers. Factor XIa initially cleaved the A alpha-chain of fibrinogen and subsequently degraded the B beta-chain. However, the cleavage sites on both chains were distinct from those susceptible to thrombin. The gamma-chain was degraded only after prolonged incubation with factor XIa. Furthermore, the profile of fibrinogen proteolysis by factor XIa was distinctly different from that of plasmin-catalyzed fibrinogenolysis. Unlike plasmin, factor XIa was not able to cleave the NH2-terminus of the B beta-chain of fibrinogen. Moreover, factor XIa, unlike plasmin, failed to hydrolyze fibrin. Further study of the proteolytic digests of fibrinogen produced by factor XIa may give additional insight into the mechanism of polymerization of this protein.

Biopolymers↗