Lack of effect of heparin on factor XIa.
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Factor XI↗
Biomedical subjects
C F Scott
Publications and source records attributed to C F Scott.
An immunotoxin composed of a monoclonal antitransferrin receptor antibody linked by a disulfide bond to the ribosome-inactivating protein gelonin: potent in vitro and in vivo effects against human tumors.
An immunoconjugate was prepared containing a disulfide linker between a murine monoclonal antibody (5E9), which recognized the human transferrin receptor, and the ribosome-inactivating protein gelonin. This immunoconjugate was found to consist of two major species, 5E9-gelonin2 and 5E9-gelonin1, and a minor species of 5E9-gelonin3 and less than 10% of either free antibody or gelonin. 5E9-gelonin was extremely toxic in vitro to human tumor cell lines expressing the 5E9 antigen, including a Burkitt's lymphoma, an adult T-cell acute lymphocytic leukemia, an acute myelogenous leukemia, a promyelocytic leukemia, and a cervical carcinoma line. A 24-hour exposure to 10(-9) M immunoconjugate killed 90-99.9% of tumor cells, depending on the cell line. A 5E9-negative murine leukemia was not sensitive to this conjugate. Pharmacokinetic analysis of the disappearance of this immunoconjugate from the murine circulation revealed that it had a biphasic clearance, with an initial rapid phase with a half-life (t1/2) of 3 hours and a later, slower phase with a t1/2 of about 1 day. Analysis of blood samples by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis revealed that a substantial degree of disulfide-linker breakdown occurred in vivo and that the 5E9-gelonin2 species was cleared more rapidly than the 5E9-gelonin1. With use of the same clonogenic assays used to measure in vitro toxicity, biologically active immunoconjugate could be detected in murine plasma for up to 24 hours after iv administration, but the concentration of immunoconjugate by this measure was considerably less than that predicted by SDS-gel electrophoresis. The ability to deliver immunoconjugate to tumor cells in vivo was studied with use of the Burkitt's lymphoma Namalwa as a xenograft in nude mice. It was possible to deliver substantial amounts of immunoconjugate to Namalwa cells in xenografted ascites with direct ip inoculation; lower but significant amounts of immunoconjugate could be delivered to this xenograft after systemic iv administration, provided the tumor burden was low. The 5E9-gelonin conjugate, when administered iv at the time of ip tumor inoculation, prolonged survival of nude mice bearing Namalwa or other human tumors as ascites xenografts and delayed or prevented the growth of subcutaneous nodules of Namalwa in an antigen-specific fashion after a single iv injection. Direct intratumoral administration also inhibited the growth of visible subcutaneous nodules of Namalwa. This immunoconjugate may be useful in the treatment of human cancer.
Animals↗
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↗
T cell development in B cell-deficient mice. IV. The role of B cells as antigen-presenting cells in vivo.
B cell-deficient, rabbit anti-mouse IgM-treated mice were compared with normal or normal rabbit immunoglobulin-treated controls in their ability to develop proliferative T cell responses, delayed hypersensitivity, and primary or secondary cytotoxic T cell responses. Immunization with hapten-coupled autologous spleen cells resulted in anti-mu-treated mice generating only marginal T cell responses. This decreased responsiveness was shown to be attributable not to an intrinsic T cell defect or to changes in the ability of macrophages from anti-mu-treated mice to present soluble antigen, but rather to the greatly diminished capacity of B cell-deficient spleen cells to present antigen. The results support the concept that B cells play a significant role in antigen presentation required for T cell activation.
Animals↗
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↗
Alpha-1-antitrypsin-Pittsburgh. A potent inhibitor of human plasma factor XIa, kallikrein, and factor XIIf.
Alpha-1-antitrypsin-Pittsburgh is a human variant that resulted from a point mutation in the plasma protease inhibitor, alpha 1-antitrypsin (358 Met----Arg). This defect in the alpha 1-antitrypsin molecule causes it to have greatly diminished anti-elastase activity but markedly increased antithrombin activity. In this report, we demonstrate that this variant protein also has greatly increased inhibitory activity towards the arginine-specific enzymes of the contact system of plasma proteolysis (Factor XIa, kallikrein, and Factor XIIf), in contrast to normal alpha 1-antitrypsin, which has modest to no inhibitory activity towards these enzymes. We determined the second-order-inactivation rate constant (k'') of purified, human Factor XIa by purified alpha 1-antitrypsin-Pittsburgh and found it to be 5.1 X 10(5) M-1 s-1 (23 degrees C), which is a 7,700-fold increase over the k'' for Factor XIa by its major inhibitor, normal purified alpha 1-antitrypsin (i.e., 6.6 X 10(1) M-1 s-1). Human plasma kallikrein, which is poorly inhibited by alpha 1-antitrypsin (k'' = 4.2 M-1 s-1), exhibited a k'' for alpha 1-antitrypsin-Pittsburgh of 8.9 X 10(4) M-1 s-1 (a 21,000-fold increase), making it a more efficient inhibitor than either of the naturally occurring major inhibitors of kallikrein (C-1-inhibitor and alpha 2-macroglobulin). Factor XIIf, which is not inhibited by normal alpha 1-antitrypsin, displayed a k'' for alpha 1-antitrypsin-Pittsburgh of 2.5 X 10(4) M-1 s-1. This enhanced inhibitory activity is similar to the effect of alpha 1-antitrypsin-Pittsburgh that has been reported for thrombin. In addition to its potential as an anticoagulant, this recently cloned protein may prove to be clinically valuable in the management of septic shock, hereditary angioedema, or other syndromes involving activation of the surface-mediated plasma proteolytic system.
Factor IX↗
High molecular weight kininogen is an inhibitor of platelet calpain.
Recent studies from our laboratory indicate that a high concentration of platelet-derived calcium-activated cysteine protease (calpain) can cleave high molecular weight kininogen (HMWK). On immunodiffusion and immunoblot, antiserum directed to the heavy chain of HMWK showed immunochemical identity with alpha-cysteine protease inhibitor--a major plasma inhibitor of tissue calpains. Studies were then initiated to determine whether purified or plasma HMWK was also an inhibitor of platelet calpain. Purified alpha-cysteine protease inhibitor, alpha-2-macroglobulin, as well as purified heavy chain of HMWK or HMWK itself inhibited purified platelet calpain. Kinetic analysis revealed that HMWK inhibited platelet calpain noncompetitively (Ki approximately equal to 5 nM). Incubation of platelet calpain with HMWK, alpha-2-macroglobulin, purified heavy chain of HMWK, or purified alpha-cysteine protease inhibitor under similar conditions resulted in an IC50 of 36, 500, 700, and 1,700 nM, respectively. The contribution of these proteins in plasma towards the inhibition of platelet calpain was investigated next. Normal plasma contained a protein that conferred a five to sixfold greater IC50 of purified platelet calpain than plasma deficient in either HMWK or total kininogen. Reconstitution of total kininogen deficient plasma with purified HMWK to normal levels (0.67 microM) completely corrected the subnormal inhibitory activity. However, reconstitution of HMWK deficient plasma to normal levels of low molecular weight kininogen (2.4 microM) did not fully correct the subnormal calpain inhibitory capacity of this plasma. These studies indicate that HMWK is a potent inhibitor as well as a substrate of platelet calpain and that the plasma and cellular kininogens may function as regulators of cytosolic, calcium-activated cysteine proteases.
Blood Physiological Phenomena↗
Role of arginine residues in the coagulant activity of high molecular weight kininogen.
High molecular weight (HMW) kininogen, the cofactor for activation of the contact system of plasma proteolysis, transports and optimally positions prekallikrein and factor XI on a negatively charged surface, allowing those zymogens to be activated by surface-bound factor XIIa. HMW kininogen circulates in plasma as a procofactor that, after cleavage by kallikrein or factor XIIa, gains ability to bind to the surface. The mechanism responsible for this increased affinity for the surface is unknown. We hypothesized that modification of arginine residues may prevent cleavage of HMW kininogen, since the initial kallikrein-induced cleavage sites on the HMW kininogen molecule are at the NH2 terminal and the COOH terminal of the bradykinin-containing portion of the molecule, each of which contains arginine. We found that modification with butanedione of four arginine residues in the HMW kininogen molecule prevented bradykinin release, which results from cleavage of HMW kininogen. Furthermore, HMW kininogen coagulant activity was lost, in proportion to the degree of arginine modification, until 6.6 residues had been modified. Complex formation with prekallikrein, however, was found to be uneffected by the modification of modified HMW kininogen. To account for the loss of coagulant activity, we also examined the ability of modified HMWKa (active cofactor) to bind to an activating surface. The affinity of modified HMWKa for kaolin was tenfold less than the affinity of unmodified HMWKa. These data suggest that arginine residues play a critical role in the ability of HMW kininogen to function as an activation cofactor, both by preventing the cleavages that produce HMWKa as well as by decreasing the affinity of HMWKa for the surface.
Arginine↗
Cleavage of human high molecular weight kininogen by factor XIa in vitro. Effect on structure and function.
We have recently demonstrated that human high molecular weight kininogen (HMWK) is a pro-cofactor that is cleaved by kallikrein to yield a two-chain cofactor (HMWKa) and the nanopeptide bradykinin. This proteolysis enhances its association with an activating surface, an event necessary for expression of its cofactor activity. We now report that factor XIa is capable of hydrolyzing HMWK and releasing bradykinin in a purified system as well as cleaving and inactivating HMWK in a plasma environment during the contact-activation process. The profile of proteolysis differs from that produced by kallikrein and by factor XIIa in that the first cleavage by factor XIa yields 75- and 45-kDa polypeptides, whereas both factor XIIa and kallikrein initially produce 65- and 56-kDa species. Further proteolysis by all three enzymes eventually produces similar heavy chains (Mr = 65,000) and light chains (Mr = 45,000). However, the amount of factor XIa generated in plasma during contact activation further degrades the light chain of HMWK, eventually destroying its coagulant activity. Furthermore, in a purified system, enhancement of the degradation of HMWK coagulant activity by factor XIa was achieved when kallikrein was included in the incubation mixture, suggesting that the preferred substrate for factor XIa is the active form of HMWK (HMWKa), and not the pro-cofactor. These data suggest that factor XIa has the potential to act as a regulator of contact-activated coagulation by virtue of its ability to destroy the cofactor function of HMWK after its generation by either kallikrein, factor XIIa, or to a lesser extent, factor XIa, itself.
Bradykinin↗
Regulation of hapten-specific T-cell response. II. Functional analysis of helper T cells and cytotoxic T cells in animals suppressed by azobenzenearsonate (ABA)-specific suppressor T cells.
The administration of azobenzenearsonate-modified syngeneic spleen cells (ABA-SC) intravenously induces a population of first order hapten-specific inducer suppressor T cells (Ts1), which downregulate various aspects of T-cell-mediated immune responses via a well defined suppressor-T-cell pathway. In this study, we investigated the effects of these suppressor cells on the generation of ABA-specific cytolytic T lymphocytes (CTL) and helper T cells (Th) in vivo. We found evidence for functional impairment of ABA-activated Th and ABA-specific CTL precursors (CTLp) in the suppressed animals by a number of different in vitro criteria. Functional analysis of ABA-specific CTLp and ABA-activated Th in suppressed animals revealed that ABA-specific Ts inhibit the generation of CTL by impairing the antigen-specific activation of Th, which may in turn, prevent the clonal expansion of antigen-specific CTLp. The significance of these findings in relationship to our understanding of the cellular interactions necessary for the generation of CTL and the mode of action and mechanisms of suppressor T cells is discussed.
Animals↗
Effect of cleavage of the heavy chain of human plasma kallikrein on its functional properties.
Human plasma kallikrein consists of an N-terminal heavy chain of molecular weight (mol wt) 52,000, linked by disulfide bonds to two light chain variants (mol wt 36,000 or 33,000). Although the active catalytic site of kallikrein resides on the C-terminal light chain, the role of the N-terminal heavy chain is less clear. We therefore studied an enzyme designated beta-kallikrein, containing a single cleavage in the heavy chain (mol wt 28,000 + 18,000) and compared it to the enzyme, alpha-kallikrein, with an intact heavy chain. The rates of inactivation by C1 inhibitor of plasma alpha- and beta-kallikreins were kinetically identical, as measured by residual amidolytic activity, after various times of incubation with the inhibitor. Both enzymes reacted completely with C1 inhibitor after 18 hours and formed identical C1 inhibitor-kallikrein complexes of mol wt 195,000. The rate of activation of factor XII by alpha-kallikrein and beta-kallikrein was similar. In contrast, the rate of cleavage of high molecular weight kininogen (HMWK) by alpha-kallikrein was at least fivefold faster and the ratio of coagulant activity to amidolytic activity was fourfold greater than for beta-kallikrein. Plasma alpha-kallikrein, at concentrations potentially achievable in plasma, induced aggregation of neutrophils, but beta-kallikrein failed to elicit this response. In addition, human neutrophils pretreated with cytochalasin B released 2.46 +/- 0.10 microgram/10(7) cells of elastase antigen, but beta-kallikrein released only 0.25 +/- 0.10 micrograms/10(7) cells. These observations suggest that cleavage of the heavy chain influences the rate of cleavage of HMWK and decreases its coagulant activity. Moreover, an intact heavy chain appears to be requisite to support the ability of kallikrein to aggregate neutrophils and release elastase.
Blood Coagulation Tests↗
Alpha 1-antitrypsin-Pittsburgh: a potent inhibitor of human plasma factor XIa, kallikrein, and factor XIIf.
AT-P is a potent inhibitor of factor XIa. It has a greater affinity for factor XIa (5.1 X 10(5) M-1 sec-1), than it does for thrombin (3.1 X 10(5) M-1 sec-1). This recently cloned protein may be clinically valuable in the management of disorders involving activation of the contact system of plasma proteolysis.
Factor XI↗
Cleavage of human high molecular weight kininogen markedly enhances its coagulant activity. Evidence that this molecule exists as a procofactor.
High molecular weight kininogen (HMW)-kininogen, the cofactor of contact-activated blood coagulation, accelerates the activation of Factor XII, prekallikrein, and Factor XI on a negatively charged surface. Although prekallikrein and Factor XI circulate as a complex with HMW-kininogen, no physical association has been demonstrated between Factor XII and HMW-kininogen, nor has the order of adsorption to surfaces of these proteins been fully clarified. In this report we explore the requirements for adsorption of HMW-kininogen to a clot-promoting surface (kaolin), in purified systems, as well as in normal plasma and plasma genetically deficient in each of the proteins of the contact system. The fraction of each coagulant protein associated with the kaolin pellet was determined by measuring the difference in coagulant activity between the initial sample and supernatants after incubation with kaolin, or by directly quantifying the amount of 125I-HMW-kininogen that was associated with the kaolin pellet. In normal plasma, the adsorption of HMW-kininogen to kaolin increased as the quantity of kaolin was increased in the incubation mixture. However, the HMW-kininogen in Factor XII-deficient plasma did not absorb appreciably to kaolin. Furthermore, the quantity of HMW-kininogen from prekallikrein-deficient plasma that adsorbed to kaolin was decreased as compared with normal plasma. These observations suggested that HMW-kininogen in plasma must be altered by a reaction involving both Factor XII and prekallikrein in order for HMW-kininogen to adsorb to kaolin, and to express its coagulant activity. Subsequently, the consequence of the inability of HMW-kininogen to associate with a negatively charged surface results in decreased surface activation. This assessment was derived from the further observation of the lack of prekallikrein adsorption and the diminished Factor XI adsorption in both Factor XII-deficient and HMW-kininogen-deficient plasmas, since these two zymogens (prekallikrein and Factor XI) are transported to a negatively charged surface in complex with HMW-kininogen. The percentage of HMW-kininogen coagulant activity that adsorbed to kaolin closely correlated (r = 0.98, slope = 0.97) with the amount of 125I-HMW-kininogen adsorbed, suggesting that adsorption of HMW-kininogen results in the expression of its coagulant activity. Since kallikrein, which is known to cleave HMW-kininogen, is generated when kaolin is added to plasma, we tested the hypothesis that proteolysis by kallikrein was responsible for the enhanced adsorption of HMW-kininogen to kaolin. When purified HMW-kininogen was incubated with purified kallikrein, its ability to absorb to kaolin increased with time of digestion until a maximum was reached. Moreover, (125)I-HMW-kininogen, after cleavage by kallikrein, had markedly increased affinity for kaolin than the uncleaved starting material. Furthermore, fibrinogen, at plasma concentration (3 mg/ml), markedly curtailed the adsorption of a mixture of cleaved and uncleaved HMW-kininogen to kaolin, but was unable to prevent fully cleaved HMW-kininogen from adsorbing to the kaolin. Addition of purified kallikrein to Factor XII-deficient plasma, which bypasses Factor XII-dependent contact-activation amplified the ability of its HMW-kininogen to adsorb to kaolin. These observations indicate that HMW-kininogen is a procofactor that is activated by kallikrein, a product of a reaction which it accelerates. This cleavage, which enhances its association with a clot-promoting surface in a plasma environment, is an event that is necessary for expression of its cofactor activity. These interactions would allow coordination of HMW-kininogen adsorption with the adsorption of Factor XII, which adsorbs independently of cleavage, to the same negatively charged surface.
Adsorption↗
Comparison of antigen-specific T cell responses in autoimmune MRL/Mp-lpr/lpr and MRL/Mp-+/+ mice.
The MRL-1 mouse develops severe autoimmune disease characterized by high titers of autoantibodies at an early age (3 to 5 mo). The congeneic MRL-n mouse, which differs only in the lymphoproliferative (lpr) gene, exhibits no such pathologic or serologic abnormalities at the same age. We examined antigen-specific T cell responses in the MRL-1 mouse and compared them to age- and sex-matched MRL-n controls. We found broad defects in these responses in the MRL-1 mouse; an inability to generate primary allospecific and hapten-specific cytolytic T lymphocytes (CTL), secondary hapten- and virus-specific CTL, as well as a deficient proliferative response to hapten and natural antigens and a weak delayed-type hypersensitivity response were demonstrated. Our data furthermore suggest a lack of interleukin 2 (IL 2) acceptor sites in the proliferating T cell, while suggesting no such lack on CTL precursors. In fact, the deficient CTL responses in MRL-1 mice can be restored to levels seen in MRL-n by the in vitro addition of IL 2. The implications of these findings and the possible explanations for the relative deficit in helper function in the MRL-1 mouse are discussed.
Aging↗
Regulation of the coagulant activity and surface binding of high molecular weight kininogen.
Cleavage of the procofactor, high molecular weight kininogen, by kallikrein, is required for expression of it cofactor activity. Coagulant activity associated with the light chain of high molecular weight kininogen is destroyed by proteolysis by factor XIa. The heavy chain of high molecular weight kininogen augments the binding of the light chain to activating surfaces without itself expressing coagulant activity.
Blood Coagulation Factors↗
Amidolytic assay of human factor XI in plasma: comparison with a coagulant assay and a new rapid radioimmunoassay.
The traditional coagulant assay for plasma factor XI suffers from a relatively high coefficient of variation, the need for rare congenitally deficient plasma, and a poor correlation between precision and sensitivity. We have developed a simple functional amidolytic assay for factor XI in plasma using the chromogenic substrate PyrGlu-Pro-Arg-p-nitroanilide (S-2366). After inactivation of alpha 1-antitrypsin, CI inhibitor, and other plasma protease inhibitors with CHCI3, plasma was incubated with kaolin, in the absence of added calcium, which limited the enzymes formed to those dependent on contact activation. Soybean trypsin inhibitor was used to minimize the action of kallikrein on the substrate. Once the reaction was complete, corn trypsin inhibitor was used to inactive factor XIIa, the enzyme generated by exposure of plasma to negatively charged surfaces, which had activated the factor XI. The assay is highly specific for factor XI, since plasma totally deficient in that zymogen yielded only 1%-3% of the enzymatic activity in normal plasma under identical conditions. The requirements for complete conversion of factor XI to XIa in plasma within 60 min were, respectively, factor XII, 0.6 U/ml, and high molecular weight kininogen, 0.2 U/ml. Prekallikrein was not an absolute requirement for complete activation but did accelerate the reaction. The intraassay coefficient of variation was 3.4%, and the mean of 35 normal plasmas was 1.00 U +/- 0.24 SD. In addition, a new rapid radioimmunoassay was devised using staphylococcal protein A as the precipitating agent for a complex of factor XI antigen with monospecific rabbit antibody. The mean was 1.01 U +/- 0.30 SD. The correlation coefficients for amidolytic versus coagulant and amidolytic versus radioimmunoassay were r = 0.95 for the former and 0.96 for the latter. Thus, a simple, accurate amidolytic assay and a radioimmunoassay have been devised for measuring factor XI in plasma that correlate well with the coagulant activity of factor XI, as determined in our laboratory.
Animals↗
Prekallikrein activation and high-molecular-weight kininogen consumption in hereditary angioedema.
Patients with hereditary angioedema lack C-1 inhibitor, a plasma alpha 2-glycoprotein that inhibits both the proteolytic action of C1, the activated first component of the complement system, and the activity of components of the contact phase of coagulation: kallikrein, factor XIa, and factor XIIa. Such patients have been shown to have low levels of C4 and C2, the natural substrates for C-1, but the levels were not correlated with the presence of symptoms. We studied three patients with angioedema for evidence of activation of the contact system and found that during a symptomatic period they had decreased levels of prekallikrein, a substrate for the activated forms of factor XII, and reductions in high-molecular-weight kininogen, a substrate for plasma kallikrein. These observations suggest that zymogens of the contact system are activated during attacks of hereditary angioedema and that some of the clinical manifestations may be mediated through products of this pathway, such as kinins.
Adolescent↗
Human plasma kallikrein releases neutrophil elastase during blood coagulation.
Elastase is released from human neutrophils during the early events of blood coagulation. Human plasma kallikrein has been shown to stimulate neutrophil chemotaxis, aggregation, and oxygen consumption. Therefore, the ability of kallikrein to release neutrophil elastase was investigated. Neutrophils were isolated by dextran sedimentation, and elastase release was measured by both an enzyme-linked immunosorbent assay, and an enzymatic assay using t-butoxy-carbonyl-Ala-Ala-Pro-Val-amino methyl coumarin as the substrate. Kallikrein, 0.1-1.0 U/ml, (0.045-0.45 microM), was incubated with neutrophils that were preincubated with cytochalasin B (5 micrograms/ml). The release of elastase was found to be proportional to the kallikrein concentration. Kallikrein released a maximum of 34% of the total elastase content, as measured by solubilizing the neutrophils in the nonionic detergent Triton X-100. A series of experiments was carried out to determine if kallikrein was a major enzyme involved in neutrophil elastase release during blood coagulation. When 10 million neutrophils were incubated in 1 ml of normal plasma in the presence of 30 mM CaCl2 for 90 min, 2.75 micrograms of elastase was released. In contrast, neutrophils incubated in prekallikrein-deficient or Factor XII-deficient plasma released less than half of the elastase, as compared with normal plasma. The addition of purified prekallikrein to prekallikrein-deficient plasma restored neutrophil elastase release to normal levels. Moreover, release of elastase was enhanced in plasma deficient in C1-inhibitor, the major plasma inhibitor of kallikrein. This release was not dependent upon further steps in the coagulation pathway, or on C5a, since levels of elastase, released in Factor XI- or C5-deficient plasma, were similar to that in normal plasma, and an antibody to C5 failed to inhibit elastase release. These data suggest that kallikrein may be a major enzyme responsible for the release of elastase during blood coagulation.
Blood Coagulation↗