Michael Silverberg

Mechanisms of activation of the classical pathway of complement by Hageman factor fragment.

The mechanism by which a fragment of activated Hageman factor (HFf) activates the classical pathway of complement in serum or platelet-poor plasma has been further delineated. When serum or platelet-poor plasma was incubated with various concentrations of HFf, the total complement hemolytic activity was reduced in a dose-dependent manner. This activation appears to be due to the direct interaction of HFf with macromolecular C1, since incubation of purified C1 with HFf resulted in dissociation of the subunits with concomitant reduction of C1r antigenicity that is indicative of C1 activation. HFf-dependent activation was prevented by prior treatment of HFf with the active site-directed inhibitor, H-D-proline-phenylalanine-arginine chloromethyl ketone or with a specific inhibitor of activated HF derived from corn. Incubation of HFf with highly purified C1r also resulted in activation of C1r as assessed directly using a synthetic substrate or indirectly by activation of C1s and consumption of C2. However, incubation of HFf with highly purified C1s resulted in formation of activated C1s (C1s-) but this was less efficient than HFf activation of C1r. We therefore conclude that activation of C1 in macromolecular C1 is the result of HFf conversion of C1r to C1r; activation of C1s then occurs primarily by C-1r and to a lesser degree by the direct action of HFf.

The mechanism by which the light chain of cleaved hmw-kininogen augments the activation of prekallikrein, factor XI and hageman factor☆

Abstract HMW-kininogen binds prekallikrein and factor XI to form two bimolecular complexes in plasma; upon addition of certain negatively charged surfaces, it functions as a coagulation cofactor and facilitates the activation of Hageman factor, prekallikrein and factor XI. We have investigated the mechanism by which HMW-kininogen functions in each of these steps. HMW-kininogen was found to augment the binding of prekallikrein and factor XI to kaolin in a plasma system suggesting that the prekallikrein-HMW kininogen complex and the factor XI-HMW kininogen complex are attached to surfaces via HMW-kininogen. The attachment was shown to be mediated by the light chain derived from cleaved HMW-kininogen and is consistent with previous data demonstrating that the light chain is the coagulant part of the molecule. Prekallikrein can be readily activated when bound to the surface by this HMW-kininogen linkage; however, when an equal quantity of prekallikrein was bound directly to the surface, it was not activatable even after the addition of HMW-kininogen. Thus, binding of prekallikrein (and factor XI) to the surface via HMW-kininogen appears to place them in a conformationally favorable position for activation by activated Hageman factor. This appears to be the major function of HMW-kininogen as a coagulation cofactor. In addition, HMW-kininogen attachment of prekallikrein (kallikrein) to the surface is a reversible interaction which is not the case when direct binding to the surface occurs. In this fashion, a portion of the kallikrein formed can dissociate and interact with other Hageman factor molecules. These data suggest that the effect of HMW-kininogen upon Hageman factor activation is indirect and acts to increase the effective concentration of kallikrein available for Hageman factor cleavage.

Autoactivatability of human Hageman factor (factor XII)

Abstract Purified Hageman factor was found to autodigest upon binding to a negatively charged surface such as kaolin. Assessment by incorporation of tritiated diisopropylfluorophosphate indicated that this cleavage was accompanied by activation and that the two known forms of activated Hageman factor result. Cleavage within a critical disulfide bridge generated activated Hageman factor, a two-chain enzyme of molecular weight 80,000 as well as the active Hageman factor fragment, a 28,000 molecular weight cleavage product. The autocleavage seen was dependent upon the percentage of activated Hageman factor in the starting material and was independent of HMW-kininogen. This result suggest that initiation of the intrinsic coagulation cascade may, in part, depend upon the autoactivatability of Hageman factor described herein. This observation may in turn, account for the ability of prekallikrein deficient plasma to gradually autoactivate as a function of the time of contact with initiating surfaces.

The Activation of the Contact System of Human Plasma by Polysaccharide Sulfates

Blood drawn into a clean glass tube will rapidly clot, yet if a plastic or siliconized tube is used, clotting is delayed. This phenomenon of “contact activation” is mediated by a group of plasma proteins, the contact system, whose interaction with a variety of negatively charged surfaces results in initiation of the intrinsic coagulation pathway.lS2 These proteins also mediate the factor-XII-dependent pathways of kinin formation and fibrinolysis. The paradox of contact activation is that the congenital absence of any of the proteins of the contact system does not lead to overt clinical deficiencies, although the effect on the in vitro clotting assay is severe. However, there is increasing evidence that activation in vivo occurs in response to several situations including hereditary angioedema and gram negative sepsis. Also, blood or plasma in contact with nonphysiological surfaces may generate significant quantities of bradykinin if contact activation occurs. Thus, the mechanism of the initiation of contact activation is a subject of considerable interest.

MECHANISMS FOR HAGEMAN FACTOR ACTIVATION AND ROLE OF HMW KININOGEN AS A COAGULATION COFACTOR

Our present concept of the initiating reactions of the intrinsic coagulation pathway is outlined in Figure 5. Although we remain unsure of the etiology shown in Figures 3 and 4, the major function of HMW kininogen is to bind prekallikrein and factor XI in plasma and attach them to surfaces in a conformation that allows activation by HFa. The HMW kininogen--dependent augmentation of the binding of prekallikrein and factor XI to the surface that is seen in plasma (but not buffer systems) would appear to be of lesser importance. Once activated, however, dissociation of kallikrein from the surface allows it to attack adjacent Hageman factor molecules on the same or other particles; this reaction appears to be more rapid than the rate of Hageman factor autoactivation. Thus, the rapid burst of HFa formation seen in normal plasma is kallikrein dependent. It is also dependent upon HMW kininogen, but this appears to be an indirect relationship. The HMW kininogen augments the amount of prekallikrein bound, allows activation to kallikrein, and is needed for kallikrein dissociation from the surface. These three effects all yield a marked increase in the effective ratio of kallikrein/Hageman factor at the surface-fluid interface, and this may be the condition required for rapid HFa formation.

Effect of neurotropin® on the activation of the plasma kallikrein-kinin system

Bradykinin (BK), an important mediator of allergic reactions and pain induction, is released by the activation of the plasma kallikrein-kinin (K-K) cascade. Neurotropin is a biological material obtained from inflamed rabbit skin inoculated with vaccinia virus and is widely used clinically in Japan as an effective agent for these disorders. Since its mechanism of action is not clearly known, we have investigated the effects of Neurotropin on the human plasma K-K system. In dextran sulfate-activated plasma, Neurotropin inhibited the formation of BK, the cleavage of high molecular weight kininogen (HK) and the formation of kallikrein-C1 inhibitor and activated coagulation factor XII (FXIIa)-C1 inhibitor complexes. Experiments using purified enzyme of the K-K cascade indicated that Neurotropin inhibited surface-mediated activation of coagulation factor XII (FXII) and the activation of prekallikrein by FXIIa. Neurotropin also inhibited the binding of FXII and HK to the activating surface. These data suggest that the ameliorating effects of Neurotropin in allergic disorders and pain syndromes may be related to this ability to inhibit activation of the K-K cascade and consequently the formation of BK.

Assembly of the Human Plasma Kinin-Forming Cascade along the Surface of Vascular Endothelial Cells

The generation of bradykinin by contact activation requires autoactivation of factor XII (Hageman factor) upon initiating surfaces, conversion of prekallikrein to kallikrein, and digestion of high-molecular-weight (HMW) kininogen. Endothelial cells have a high-affinity receptor that binds either HMW kininogen or factor XII in a zinc-dependent interaction, and activation of factor XII can occur along this surface to initiate kinin formation. Tissue injury, exposure of proteoglycans, or release of mast cell heparin will markedly accelerate these reactions. The bradykinin released binds to endothelial cell B-2 receptors along the inner surface of blood vessels which results in dilatation and increased vascular permeability.

Hageman Factor Activation by Polysaccharides: Effect of Molecular Weight

Hageman factor dependent generation of bradykinin in human plasma is initiated by the contact of human plasma with substances bearing fixed negative charges1 2. Activation occurs in vivo in several situations including hereditary angioedema and gram negative sepsis. Also, blood or plasma in contact with nonphysiological surfaces may generate significant quantities of bradykinin if contact activation occurs. The proteins involved in the initial reactions at the surface are Hageman factor (factor XII), prekallikrein and high molecular weight kininogen. Both prekallikrein and Hageman factor circulate as zymogens but are converted to active proteases upon contact with a surface in the presence of high molecular weight kininogen3.

Quantitative assays of pharmacologic aerosols used in studying asthma

To more nearly accurately quantitate the dose of pharmacologic agents delivered to human and animal airways via aerosols, we have developed a monodisperse aerosol containing either methacholine or histamine that permits a light scattering device (tyndallometry) to measure accurately the quantity of inspired and expired particles. These aerosols (described in previous studies) are simultaneously tagged with a radioactive label (technetium 99m) to permit the use of external gamma camera imaging. Present work focuses on the development of assay techniques to measure the quantity of methacholine delivered in these aerosols. The lack of specific radioimmune or radioenzyme assays coupled with the cross-reaction of organic contaminants with conventional chemical reagents for measuring methacholine required the development of separative techniques to isolate the methacholine from the organic aerosol contaminants. With aqueous extraction and column separation we have been able to completely isolate the methacholine from these contaminants. This allows the application of standard spectrophotometric assays for methacholine to quantitate the methacholine in the resulting solution. These separative techniques will permit the use of these aerosols in quantitative studies of airway reactivity.