RESUMEN
Purified domains of low molecular weight kininogen (LK) can be used directly to determine the epitopes of monoclonal antibodies (mAbs) that have been shown to influence kininogen function. LK, purified from plasma by carboxymethyl-papain-Sepharose 4B affinity chromatography and kaolin adsorption, was digested by trypsin and chymotrypsin. The domains of LK were then separated by gel filtration followed by carboxymethyl-papain-Sepharose 4B affinity chromatography. Using the purified domains of LK's heavy chain, the regions on kininogens' heavy chain which various monoclonal antibodies are directed to were determined by enzyme-linked immunosorbent assay and immunoblotting. MAb 2B5 which neutralized kininogens' ability to inhibit calpain cross-reacted with domains 2 and 3. MAb HKH8 which reacted with kininogens' domain 1 and 2 was found to inhibit 125I-HK binding to platelets. At two-fold molar excess, mAb HKH8 was a better inhibitor of 125I-HK binding to platelets than higher concentrations, where the antibody was shown to cause increased binding to platelets. Alternatively, HKH8 F(ab')2 completely inhibited 125I-HK binding to platelets even at high concentrations of antibody. These studies indicate that purified domains of kininogens' heavy chain can be used to rapidly localize epitopes for antibodies. Further, mAb HKH8 should be a valuable probe to understand the mechanisms of kininogens' binding to platelets.
Asunto(s)
Anticuerpos Monoclonales , Plaquetas/metabolismo , Quininógenos/inmunología , Quininógenos/metabolismo , Adsorción , Ensayo de Inmunoadsorción Enzimática , Humanos , Immunoblotting , Caolín , Cinética , Quininógenos/química , Peso Molecular , Conformación ProteicaRESUMEN
The unstimulated platelet surface contains a specific and saturable binding site for high molecular weight kininogen (HK) and low molecular weight kininogen (LK). Investigations were performed with purified heavy and light chains of HK to determine which portion(s) of the HK molecule binds to the platelet surface. Purified 64-Kd heavy chain of HK and 56-Kd light chain of HK, independently, inhibited 125I-HK binding to unstimulated platelets with a 50% inhibitory concentration (IC50) of 84 nmol/L (apparent Ki, 30 nmol/L) and 30 nmol/L (apparent Ki, 11 nM), respectively. The ability of each of the purified chains of HK to independently inhibit 125I-HK binding was not due to cleavage, reduction, and alkylation of the protein, because two-chain HK, produced by treating HK the same way as purifying the separate chains, inhibited binding similarly to intact HK. Further, purified LK alone inhibited 125I-HK binding to platelets (Ki, 17 +/- 1 nmol/L, n = 7). The 64-Kd heavy chain of HK was a competitive inhibitor on a reciprocal plot of 125I-HK-platelet binding with an apparent Ki of 28 +/- 6 nmol/L (n = 4). Independently, purified 56-Kd light chain of HK was also found to be a competitive inhibitor of 125I-HK-platelet binding, with an apparent Ki of 11 +/- 3 nmol/L (mean +/- SEM, n = 4). These indirect studies indicated that HK binds to platelets by two portions of the molecule, one on the heavy chain and another on the light chain. Studies with 125I-light chain of HK showed that it specifically bound directly to platelets in the presence of zinc, since it was blocked by HK, light chain of HK, or EDTA, but not by LK, C1s, C1 inhibitor, plasmin, factor XIII, or fibrinogen. Purified light chain of HK did not inhibit direct 125I-LK binding to platelets. HK was found to bind to platelets in an unmodified form. HK bound to platelets was cleaved by plasma or urinary kallikrein at a slower rate than the same concentration of soluble HK or HK bound and subsequently eluted from the platelet surface. Cleavage of platelet-bound HK correlated with bradykinin liberation. These studies indicate that HK has two domains on its molecule that bind to platelets. Further, platelet-bound HK is protected from kallikreins' proteolysis. This latter finding suggests that cell binding may modify the rate of bradykinin liberation from HK.
Asunto(s)
Plaquetas/metabolismo , Calicreínas/metabolismo , Quininógenos/metabolismo , Adulto , Secuencia de Aminoácidos , Unión Competitiva , Femenino , Humanos , Immunoblotting , Calicreínas/orina , Quininógenos/antagonistas & inhibidores , Quininógenos/química , Masculino , Datos de Secuencia Molecular , Peso Molecular , Oligopéptidos/química , Oligopéptidos/farmacología , Proteínas Recombinantes/metabolismoRESUMEN
Hereditary angioedema (HAE), an autosomal disorder caused by a deficiency of C1 inhibitor, is characterized by attacks of localized swelling, laryngeal edema, or abdominal pain. Plasma samples from one pregnant patient were studied serially by functional and quantitative immunochemical assays as well as immunoblot assays for high molecular weight kininogen (HMWK) and/or prekallikrein/kallikrein (PK/K). An immunoblot of this patient's HMWK from plasma obtained before she became pregnant and when she was well revealed that it was mostly an intact protein of 120 kd, similar to immunoblot results of normal plasma HMWK. In plasma samples taken throughout her pregnancy, before, during, and after clinical attacks of angioedema, all of her plasma HMWK was shown to be cleaved into the 45 kd light chain form. After delivery of the infant the 120 kd form of intact plasma HMWK returned to her plasma. In comparison, immunoblot studies on 21 normal and abnormal pregnancies revealed that plasma HMWK was an intact protein at 120 kd. That this patient's plasma during her pregnancy was contact activated was determined by additional immunoblot studies for PK/K. Immunoblot assay for plasma PK/K revealed kallikrein-alpha 2-macroglobulin complexes and a 50 kd PK/K form seen only in activated plasma samples. The findings of kallikrein-alpha 2-macroglobulin complexes and a 50 kd PK/K form disappeared after delivery. These combined studies on this patient show that the structures of HMWK and prekallikrein as indicated by immunoblot assays were altered during pregnancy. Immunoblot assays for detection of changes in the structure of HMWK and prekallikrein may be objective laboratory studies for documenting clinical attacks of hereditary angioedema, their onset, and their resolution.