Robin A. Pixley

The contact system contributes to hypotension but not disseminated intravascular coagulation in lethal bacteremia. In vivo use of a monoclonal anti-factor XII antibody to block contact activation in baboons.

The hypotension and disseminated intravascular coagulation (DIC) in bacteremia is thought to be mediated by the combined actions of cytokines, prostaglandins, and complement. The contact system, via the release of bradykinin and the activation of Factor XI, has been postulated to be contributing to the observed hypotension and DIC. Using a mAb to Factor XII (C6B7), we blocked the activation of the contact system in an established experimental baboon model in which Escherichia coli was infused to produce lethal bacteremia with hypotension. The untreated group (n = 5) displayed contact activation, manifested by a significant decrease in high molecular weight kininogen (HK) and a significant increase in alpha 2 macroglobulin-kallikrein complexes (alpha 2M-Kal). The C6B7-treated group (n = 5) showed an inactivation of Factor XII and the changes in HK and alpha 2M-Kal complexes were prevented. Both groups developed DIC manifested by a decrease in platelet, fibrinogen, and Factor V levels. The untreated group developed irreversible hypotension. The treated group experienced an initial hypotension that was reversed and extended the life of the animals. This study suggests that irreversible hypotension correlates with prolonged activation of the contact system, and specific antibody therapy can modulate both the pathophysiological and biochemical changes.

Binding of high molecular weight kininogen to human endothelial cells is mediated via a site within domains 2 and 3 of the urokinase receptor.

The urokinase receptor (uPAR) binds urokinase-type plasminogen activator (u-PA) through specific interactions with uPAR domain 1, and vitronectin through interactions with a site within uPAR domains 2 and 3. These interactions promote the expression of cell surface plasminogen activator activity and cellular adhesion to vitronectin, respectively. High molecular weight kininogen (HK) also stimulates the expression of cell surface plasminogen activator activity through its ability to serve as an acquired receptor for prekallikrein, which, after its activation, may directly activate prourokinase. Here, we report that binding of the cleaved form of HK (HKa) to human umbilical vein endothelial cells (HUVEC) is mediated through zinc-dependent interactions with uPAR. These occur through a site within uPAR domains 2 and 3, since the binding of 125I-HKa to HUVEC is inhibited by vitronectin, anti-uPAR domain 2 and 3 antibodies and soluble, recombinant uPAR (suPAR), but not by antibody 7E3, which recognizes the beta chain of the endothelial cell vitronectin receptor (integrin alphavbeta3), or fibrinogen, another alphavbeta3 ligand. We also demonstrate the formation of a zinc-dependent complex between suPAR and HKa. Interactions of HKa with endothelial cell uPAR may underlie its ability to promote kallikrein-dependent cell surface plasmin generation, and also explain, in part, its antiadhesive properties.

Human factor XII binding to the glycoprotein Ib-IX-V complex inhibits thrombin-induced platelet aggregation.

Factor XII deficiency has been postulated to be a risk factor for thrombosis suggesting that factor XII is an antithrombotic protein. The biochemical mechanism leading to this clinical observation is unknown. We have previously reported high molecular weight kininogen (HK) inhibition of thrombin-induced platelet aggregation by binding to the platelet glycoprotein (GP) Ib-IX-V complex. Although factor XII will bind to the intact platelet through GP Ibα (glycocalicin) without activation, we now report that factor XIIa (0.37 μm), but not factor XII zymogen, is required for the inhibition of thrombin-induced platelet aggregation. Factor XIIa had no significant effect on SFLLRN-induced platelet aggregation. Moreover, an antibody to the thrombin site on protease-activated receptor-1 failed to block factor XII binding to platelets. Inhibition of thrombin-induced platelet aggregation was demonstrated with factor XIIa but not with factor XII zymogen or factor XIIf, indicating that the conformational exposure of the heavy chain following proteolytic activation is required for inhibition. However, inactivation of the catalytic activity of factor XIIa did not affect the inhibition of thrombin-induced platelet aggregation. Factor XII showed displacement of biotin-labeled HK (30 nm) binding to gel-filtered platelets and, at concentrations of 50 nm, was able to block 50% of the HK binding, suggesting involvement of the GP Ib complex. Antibodies to GP Ib and GP IX, which inhibited HK binding to platelets, did not block factor XII binding. However, using a biosensor, which monitors protein-protein interactions, both HK and factor XII bind to GP Ibα. Factor XII may serve to regulate thrombin binding to the GP Ib receptor by co-localizing with HK, to control the extent of platelet aggregation in vivo.

Inhibition of angiogenesis by antibody blocking the action of proangiogenic high-molecular-weight kininogen.

Summary.  Previously we demonstrated that domain 5 (D5) of high‐molecular‐weight kininogen (HK) inhibits neovascularization in the chicken chorioallantoic membrane (CAM) assay and further found that kallikrein cleaved HK (HKa) inhibited FGF2‐and VEGF‐induced neovascularization, and thus was antiangiogenic. In this study, we sought to demonstrate whether uncleaved HK stimulates neovascularization and thus is proangiogenic. The chick chorioallantoic membrane was used as an in ovo assay of angiogenesis. Low‐molecular‐weight kininogen stimulates angiogenesis, indicating that D5 is not involved. Bradykinin stimulates neovascularization equally to HK and LK and is likely to be responsible for the effect of HK. A murine monoclonal antibody to HK (C11C1) also recognizes a similar component in chicken plasma as detected by surface plasmon resonance. Angiogenesis induced by FGF2 and VEGF is inhibited by this monoclonal antibody and is a more potent inhibitor of neovascularization induced by VEGF than an integrin αvβ3 antibody (LM 609). Our postulate that C11C1 inhibits the stimulation of angiogenesis by HK was confirmed when either C11C1 or D5 completely inhibited angiogenesis in the CAM induced by HK. Growth of human fibrosarcoma (HT‐1080) on the CAM was inhibited by GST‐D5 and C11C1. These results indicate HK is proangiogenic probably by releasing bradykinin and that a monoclonal antibody directed to HK could serve as an antiangiogenic agent with a potential for inhibiting tumor angiogenesis and other angiogenesis‐mediated disorders.

Regulation of leukocyte recruitment by polypeptides derived from high molecular weight kininogen

Proteolytic cleavage of single‐chain, high molecular weight kininogen (HK) by kallikrein releases the short‐lived vasodilator bradykinin and leaves behind a two‐chain, high molecular weight kininogen (HKa) reported to bind to the β2‐integrin Mac‐1 (CR3, CD11b/CD18, αMβ2) on neutrophils and exert antiad‐hesive properties by binding to the urokinase receptor (uPAR) and vitronectin. We define the molecular mechanisms for the antiadhesive effects of HK related to disruption of β2‐integrin‐mediated cellular interactions in vitro and in vivo. In a purified system, HK and HKa inhibited the binding of soluble fibrinogen and ICAM‐1 to immobilized Mac‐1, but not the binding of ICAM‐1 to immobilized LFA‐1 (CD11a/CD18, αLβ2). This inhibitory effect could be attributed to HK domain 5 and to a lesser degree to HK domain 3, consistent with the requirement of both domains for binding to Mac‐1. Accordingly, HK, HKa, and domain 5 inhibited the adhesion of Mac‐1 but not LFA‐1‐transfected K562 human erythroleukemic cells to ICAM‐1. Moreover, adhesion of human monocytic cells to fibrinogen and to human endothelial cells was blocked by HK, HKa, and domain 5. By using peptides derived from HK domain 5, the sequences including amino acids H475‐G497 (and to a lesser extent, G440‐H455) were identified as responsible for the antiadhesive effect, which was independent of uPAR. Finally, administration of domain 5 into mice, followed by induction of thioglycol‐late‐provoked peritonitis, decreased the recruitment of neutrophils by ~70% in this model of acute inflammation. Taken together, HKa (and particularly domain 5) specifically interacts with Mac‐1 but not with LFA‐1, thereby blocking Mac‐1‐dependent leukocyte adhesion to fibrinogen and endothelial cells in vitro and in vivo and serving as a novel endogenous regulator of leuko‐cyte recruitment into the inflamed tissue.—Chavakis, T., Kanse, S. M., Pixley, R. A., May, A. E., Isordia‐Salas, I., Colman, R. W., Preissner, K. T. Regulation of leukocyte recruitment by polypeptides derived from high molecular weight kininogen. FASEB J. 15, 2365–2376 (2001)

Prognostic value of assessing contact system activation and factor V in systemic inflammatory response syndrome

OBJECTIVE To test if serially sampled determinations of the contact system proteins and factor V have prognostic value for death in patients who develop the systemic inflammatory response syndrome. DESIGN Prospective, observational study with sequential measurements in an inception cohort. SETTING Medical intensive care unit (ICU) in a community hospital. PATIENTS Over a 1-yr period, a population base sample of 23 patients was selected from all ICU admissions who met established criteria for the systemic inflammatory response syndrome. INTERVENTIONS None. MEASUREMENTS AND MAIN RESULTS Components of the contact system, factor XII, prekallikrein, high-molecular-weight kininogen, factor XI, alpha 2-macroglobulin-kallikrein complexes and factor V values were measured in plasma samples collected serially (day of admission, and at 2, 12, 24, 48 and/or 72 hrs or at discharge). Data were analyzed to determine if admission values or serially obtained values within 48 hrs were useful in predicting outcome. Fourteen patients survived and nine died. At admission, in all patients, assay values indicated that prekallikrein, high-molecular-weight kininogen, and factor V were significantly lower than normal (as observed in a range of 20 to 23 healthy adults), alpha 2-macroglobulin-kallikrein complexes were higher than normal, while concentrations of factor XII and factor XI were in the normal range. No differences were detected in the admission values between survivors and nonsurvivors, nor between patients with positive or negative blood cultures. However, subsequent values demonstrated a difference in values between survivors and nonsurvivors. Survivors showed improvement in high molecular weight kininogen values and higher than normal factor V values, as compared with nonsurvivors. CONCLUSIONS Low or persistently low serial factor XII, high-molecular-weight kininogen and factor V values are associated with a poor prognosis, whereas high or increasing values of factor XII, high-molecular-weight kininogen, prekallikrein, and factor V all correlate with a favorable outcome.

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.

Effect of negatively charged activating compounds on inactivation of factor XIIa by Cl̄ inhibitor

Human factor XII, upon exposure to negatively charged surfaces such as kaolin, sulfatides, and heparin, is converted to enzymatic forms, factor XIIa and factor XIIf. Cl inhibitor has been quantitatively demonstrated to be the primary plasma inhibitor of both factor XIIa and factor XIIf. Studies were performed to determine whether the presence of artificial, negatively charged surfaces influenced the ability of Cl inhibitor to inhibit factors XIIa and XIIf. Kaolin and sulfatides slowed the rate of inhibition of factor XIIa by Cl inhibitor 4.8- and 2-fold, respectively, whereas they had no effect on the inhibition of factor XIIf by Cl inhibitor. Heparin in a concentration of 65 U/ml decreased the inhibition rate of factor XIIa by Cl inhibitor, but, at the same concentration, had less of an effect on the ability of Cl inhibitor to inhibit factor XIIf. These studies indicate that negatively charged surfaces protect factor XIIa but not factor XIIf from inhibition from Cl inhibitor. Since the difference between factors XIIa and XIIf consists of the presence of a surface binding region in factor XIIa, the basis of this protection must reside in the surface binding residues of factor XII. These in vitro events suggest that surface-bound factor XIIa may hydrolyze its physiologic substrates, factor XI and prekallikrein, in an environment partially protected from inhibition by Cl inhibitor.

Modulation of the human monocyte binding site for monomeric immunoglobulin G by activated Hageman factor.

Macrophage Fc gamma receptors play a significant role in inflammation and host defense. One monocyte/macrophage Fc gamma receptor, Fc gamma RI, the binding site for monomeric IgG, appears to be especially responsive to modulatory signals by hormones and mediators. Since Factor XIIa is generated during inflammation, we studied the effect of XIIa on Fc gamma RI. Factor XIIa, in a concentration-dependent manner (0.01-0.19 microM), reduced the number of monocyte binding sites for monomeric IgG up to 80% without altering the affinity of binding. Its precursor, Factor XII, and the low molecular weight fragment of XIIa, lacking most of the heavy chain region, did not reduce the expression of Fc gamma RI. Neither corn trypsin inhibitor (36 microM) nor diisopropylfluorophosphate (3.6 mM) diminished the effect of Factor XIIa on Fc gamma RI, although each completely inhibited the coagulant and amidolytic activity contained on the light chain of Factor XIIa. Protein synthesis was not a requirement for this effect of Factor XIIa, nor was internalization of Fc gamma RI necessary. In contrast to similar concentrations of IgG, Factor XIIa failed to displace significantly monomeric IgG from the monocyte surface, suggesting that Factor XIIa does not directly compete for Fc gamma RI. The data suggest that the heavy chain of XIIa, which contains domains that may have cell hormone activity, also contains a domain that regulates Fc gamma RI on monocytes. In addition to other hormones and mediators, Factor XIIa may serve a regulatory function in modulating Fc gamma receptor expression during inflammation.

Interactions between bradykinin (BK) and cell adhesion molecule (CAM) expression in peptidoglycan-polysaccharide (PG-PS)-induced arthritis.

Bradykinin (BK), a vasoactive, proinflammatory nonapeptide, promotes cell adhesion molecule (CAM) expression, leukocyte sequestration, inter‐endothelial gap formation, and protein extravasation in postcapillary venules. These effects are mediated by bradykinin‐1 (B1R) and‐2 (B2R) receptors. We delineated some of the mechanisms by which BK could influence chronic inflammation by altering CAM expression on leukocytes, endothelium, and synovium in joint sections of peptidoglycan‐polysaccharide‐injected Lewis rats. Blocking B1R results in significantly increased joint inflammation. Immunohistochemistry of the B1R antagonist group revealed increased leukocyte and synovial CD11b and CD54 expression and increased CD11b and CD44 endothelial expression. B2R antagonism decreased leukocyte and synovial CD44 and CD54 and endothelial CD11b expression. Although these findings implicate B2R involvement in the acute phase of inflammation by facilitating leukocyte activation (CD11b), homing (CD44), and transmigration (CD54). Treatment with a B2R antagonist did not affect the disease evolution in this model. In contrast, when both BK receptors are blocked, the aggravation of inflammation by B1R blockade is neutralized and there is no difference from the disease‐untreated model. Our findings suggest that B1R and B2R signaling show physiologic antagonism. B1R signaling suggests involvement in down‐regulation of leukocyte activation, transmigration, and homing. Further studies are needed to evaluate the B1 receptor agonists role in this model.