Substrate For Plasma Kallikrein Research Articles

Abstract P760: Plasma Kallikrein Contributes to Intracerebral Hemorrhage and Hypertension in Stroke-Prone Spontaneously Hypertensive Rats

Introduction: Hypertension is a leading risk factor for spontaneous intracerebral hemorrhage. Plasma Kallikrein (PKa) has been implicated in contributing to hemorrhage following thrombolytic therapy, however, its role in spontaneous intracerebral hemorrhage is currently not available. This report investigates the role of PKa on hemorrhage and hypertension in stroke-prone spontaneously hypertensive rats (SHRSP). Methods: SHRSP were fed with a high salt containing stroke-prone diet to increase blood pressure and induce spontaneous intracerebral hemorrhage. The roles of PKa on blood pressure, intracerebral hemorrhage, and survival in SHRSP were examined in rats receiving a PKa inhibitor (BPCCB) or plasma prekallikrein antisense oligonucleotide (PK ASO) compared with rats receiving control ASO. Effects on PKa on the proteolytic cleavage of atrial natriuretic peptide (ANP) were analyzed by tandem mass spectrometry. Results: PKa activity in plasma was increased by 29% in SHRSP on high salt diet compared with control rats. Cleaved kininogen, a substrate for PKa, was 2-fold greater in SHRSP plasma during stroke compared with SHRSP without stroke symptoms. Systemic administration of BPCCB or PK ASO to SHRSP reduced intracerebral hemorrhage (Fig. 1A) and blood pressure (Fig. 1B), and improved neurological function and survival when compared with rats receiving control ASO. Since PKa inhibition was associated with reduced blood pressure in hypertensive rats, we investigated the effects of PKa on the cleavage of ANP. Incubation of PKa with ANP resulted in the generation fragment ANP 5-28 , which displayed reduced effects on blood pressure lowering compared with full length ANP. Conclusions: PKa contributes to increased blood pressure in SHRSP, which is associated with intracerebral hemorrhage and reduced survival. PKa-mediated cleavage of ANP reduces its blood pressure lowering effects and thereby may contribute to hypertension-induced intracerebral hemorrhage.

Modulation of the Plasma Kallikrein-Kinin System Proteins Performed by Heparan Sulfate Proteoglycans.

Human plasma kallikrein-kinin system proteins are related to inflammation through bradykinin. In the proximity of its target cells, high molecular weight kininogen (H-kininogen) is the substrate of plasma kallikrein, which releases bradykinin from H-kininogen. Heparan sulfate proteoglycans (HSPGs) play a critical role in either recruiting kinin precursors from the plasma, or in the assembly of kallikrein-kinin system components on the cell surface. Furthermore, HSPGs mediate the endocytosis and activation of H-kininogen and plasma prekallikrein. In the presence of HSPGs (Chinese hamster ovary cell, CHO-K1, wild type cells) both heparin and heparan sulfate strongly inhibit the H-kininogen interaction with the cell membrane. H-kininogen is internalized in endosomal acidic vesicles in CHO-K1 but not in CHO-745 cells (mutant cells deficient in glycosaminoglycan biosynthesis). The endocytosis process is lipid raft-mediated and is dependent on caveolae. Both types of CHO cells do not internalize bradykinin-free H-kininogen. At pH 7.35, bradykinin is released from H-kininogen on the surface of CHO-745 cells only by serine proteases; however, in CHO-K1 cells either serine or cysteine proteases are found to be involved. The CHO-K1 cell lysate contains different kininogenases. Plasma prekallikrein endocytosis in CHO-K1 cells is independent of H-kininogen, and also prekallikrein is not internalized by CHO-745 cells. Plasma prekallikrein cleavage/activation is independent of glycosaminoglycans but plasma kallikrein formation is more specific on H-kininogen assembled on the cell surface through glycosaminoglycans. In this mini-review, the importance of HSPGs in the regulation of plasma kallikrein-kinin system proteins is shown.

Kininogenase activity of prostate-derived human glandular kallikrein (hK2) purified from seminal fluid.

Prostate-specific human glandular kallikrein (hK2) is an active enzyme in human seminal fluid. It is one of three serine proteases in the human kallikrein gene family, which includes hK1 (tissue kallikrein) and hK3 (prostate-specific antigen [PSA]). In order to examine kininogenase activity (i.e., production of kinin by these enzymes), we tested for bradykinin and/or Lys-bradykinin release upon incubation of hK2 and for other kallikreins with high-molecular weight kininogen (HMWK), which contains the nonapeptide bradykinin. Kinins are important regulatory peptides (especially for vascular permeability), and they may have a role in enhancing sperm motility. High-molecular weight kininogen is the substrate for plasma kallikrein (PKa potent kinin-generating enzyme circulating in blood, not of the same gene family) and for hK1. Glandular kallikrein and protein-C inhibitor (PCI)-hK2 complex, a serpin protease inhibitor that binds hK2, were purified to homogeneity by affinity and size-exclusion chromatography. About one-half of the hK2 is found in complex with PCI. The kallikrein enzymes were incubated with HMWK, and the resulting cleavage products were analyzed for kinin activity using enzyme immunoassay, high-performance liquid chromatography and mass spectrometry, and in vitro bioassay. Our results show that hK2 cleaves HMWK to produce bradykinin, not Lys-bradykinin (like hK1), and the resultant heavy (56-kDa) and light (42-kDa) chains of HMWK show similar electrophoretic mobility to those cleaved by PK. Prostate-specific antigen (hK3) had no kinin-generating activity. We also identified three other internal cleavage sites for hK2 in HMWK (Arg427, Arg437, and Arg457) that yielded two peptides, one of which is identical to a PK-cleaved peptide. Glandular kallikrein is about 500-fold less active than is PK or tissue kallikrein, but it may play a physiologically important role in bradykinin release in seminal fluid.

Biologic Activities of the Contact Factors In Vivo

Three plasma proteins, namely, Factor XII (Hageman factor), prekallikrein (Fletcher factor), and high molecular weight kininogen (Williams-Fitzgerald-Flaujeac factor), have been shown to be required for the activation of the surface-mediated pathway, or “contact system.” In vitro, such as in the activated partial thromboplastin test or on exposure to artificial surfaces, factor XII (FXII) is autoactivated to factor XIIa, which cleaves plasma prekallikrein (PK) by limited proteolysis into the active enzyme, plasma kallikrein (Fig. 1). In contrast, in vivo, high molecular weight kininogen (HK) binds to the endothelial cell surface associated with PK in a noncovalent complex. PK is activated by an endothelial cell cysteine protease to kallikrein which cleaves prourokinase to urokinase and factor XII to factor XIIa (Fig. 2). Thus, on cells, PK activation initiates the contact system, while on artificial surfaces, FXII is the initiating molecule. HK is also the substrate of plasma kallikrein, which cleaves to yield kinin-free HK (HKa) and bradykinin (Fig. 1). Although the genetically determined deficiencies of FXII, PK, and HK display abnormal in vitro plasma coagulation, none of these deficiencies is associated with a hemorrhagic diathesis. However, Factor XII, PK and HK display a wide variety of important interactions with other plasma proteins. In vitro, activated factor XII activates factor XI, factor VII and the C1 component of the complement system. Plasma kallikrein activates prourokinase (Fig. 1), prorenin, and factor XII, and cleaves HK, releasing bradykinin. Each contact protein interacts with blood and vascular cells. Factor XII downregulates a monocyte Fc receptor, releases IL-1 and IL-6 from monocytes and macrophages, and stimulates neutrophils, Plasma kallikrein activates neutrophils and potentiates fibrinolysis on endothelial cells. HK inhibits adhesion of neutrophils to artificial surfaces, enhances cellular fibrinolysis, and inhibits thrombin-induced platelet activation. Knowledge of these biochemical and cellular reactions has led to an understanding of the role of contact proteins in inflammation, fibrinolysis, cell adhesion, platelet activation and angiogenesis, and an appreciation of their participation in various pathophysiologic conditions. The molecular biology, structure and biochemical properties of FXII, PK and HK have recently been comprehensively reviewed. This review will focus on their participation in vivo in pathophysiologic processes including hypotension, inflammation, fibrinolysis, cell adhesion, angiogenesis and thrombosis.

Canine Plasma Kininogen: Evidence for the Presence of Two Kininogens and Purification of High Molecular Weight Kininogen and Characterization as Multi-Functional Molecule

The ratio of kininogen that is substrate of plasma kallikrein to kininogen, which is not substrate of plasma kallikrein in canine plasma, was about 1:3.6 by differential assay of kininogens. When the plasma was gel-filtered through a column of Sephacryl S-300 superfine, two fractions, which released kinin by trypsin, were obtained. These results indicate that two kininogens with different molecular weights are present in the plasma and they show different susceptibility to plasma kallikrein. One kininogen was purified by ion-exchange and zinc-chelating affinity chromatographies. Purified kininogen showed a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing condition and its molecular weight was 125 kDa. Released kinin from the kininogen by trypsin was bradykinin. The kininogen inhibited papain and ficin but did not inhibit bromelain at the concentration used. The kininogen bound to carboxymethylated-papain and this binding was dissociated by 3M NaSCN. Canine plasma shortened the abnormal clotting time of human high molecular weight kininogen-deficint plasma. The kininogen also shortened the abnormal clotting time of the plasma. From these results, the purified kininogen was high molecular weight kininogen and it was multi-functional protein.

Purification and characterization of a fibrinogen-clotting enzyme from the venom of jararacuçu ( Bothrops jararacussu)

A clotting enzyme of the venom of Bothrops jararacussu, denoted FC-Bj, was purified by gel chromatography on Sephadex G-100 followed by HPLC on DEAE-5PW-PAK and gel filtration on Sephacryl S-200HR. The enzyme was identified as an acidic glycoprotein which probably consists of a single polypeptide chain with isoelectric point values in the range 3.3–4.4 and containing approx. 19% carbohydrates. On polyacrylamide gel electrophoresis (PAGE) at pH 8.3, the enzyme presented a diffuse protein band. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the enzyme showed two protein bands corresponding to mol. wts of 50,600 and 60,000. After treatment of the enzyme with neuraminidase, a strongly stained band and a band weaker in staining intensity were observed on SDS-PAGE, thereby reducing the mol. wts to 44,500 and 56,300, respectively. The clotting factor possessed N- α-benzoyl-DL-arginine p-nitroanilide hydrolysing activity and coagulated fibrinogen to fibrin. These activities were 0.548 units/mg and 50.55 NIH thrombin units/mg, respectively. The proteinase was of the serine type, as indicated by sensitivity to phenylmethanesulfonyl fluoride and benzamidine. However, the amidolytic activity of this enzyme was resistant to inhibitors such as heparin, aprotinin, agmatine, EDTA, I-2581 and TLCK. The importance of disulfide bridges for the structural integrity of the purified enzyme was indicated by the loss of amidolytic activity in the presence of β-mercaptoethanol and dithiothreitol. SDS-PAGE of fibrinogen degraded with this enzyme revealed the disappearance of the Aα and Bβ chains and the appearance of lower mol. wt fragments. The enzyme was able to hydrolyse synthetic chromogenic substrates with arginine as the C-terminal residue, and the kinetic parameters were determined. It hydrolysed the plasma kallikrein substrate H-D-Pro-Phe-Arg-pNA (S-2302) and produced kinin-releasing activity causing ileum contraction. In addition, hypotension and bradycardia were observed in urethane-anesthetized rats upon i.v. injection of the enzyme.

CDNA and deduced amino acid sequence of human PK-120, a plasma kallikrein-sensitive glycoprotein

PK-120 is a substrate for plasma kallikrein (PK), recently purified from human plasma. Here we have established the cDNA sequence for human PK-120 mRNA. The deduced amino sequence of PK-120 revealed that it consists of 902 amino acid residues with a calculated mass of 116,423 Da. The putative cleavage sites by PK have been proposed, suggesting that PK-120 may be a precursor of a bioactive peptide. Most interestingly, PK-120 showed significant sequence identities to heavy chains (HCs) of the inter-α-trypsin inhibitor (ITI) superfamily.

Purification and characterization of PK-120, a novel substrate for plasma kallikrein, from guinea pig plasma.

PK-120 is a novel substrate of plasma kallikrein isolated from human plasma. Western blotting using anti-human PK-120 polyclonal antibodies demonstrated the presence of a PK-120-like protein in guinea pig, rat and mouse plasma. We purified the immunoreactive-120 kDa protein from guinea pig plasma by polyethylene glycol fractionation followed by ion-exchange chromatography using Q-Sepharose, heparin-Sepharose, Mono-Q and gel filtration with TSK G3000. The 120 kDa protein thus isolated was similar to human PK-120 with respect to limited proteolysis by kallikrein, amino acid composition, and the N-terminal amino acid sequence, indicating that the immunoreactive 120 kDa protein was guinea pig PK-120.

Purification and characterization of a novel substrate for plasma kallikrein (PK-120) in human plasma

A 120 kDa plasma protein, which is susceptible to plasma kallikrein, was purisfied from human plasma by polyethylene glycol fractionation followed by ion exchange chromatography using Q-Sepharose, S-Sepharose, and hydroxyapatite and gel filtration on Sephacryl S-200. The 120 kDa protein, termed PK-120 in this paper, was a single polypeptide chain containing about 20% sugar by weight and its concentration in plasma was estimated to be 80μg/ml by ELISA. At least three fragments, 100, 70, and 35 kDa, were produced from PK-120 by plasma kallikrein. The N-terminal sequence and Western blot demonstrated that PK-120 was first cleaved to yield the 100 and 35 kDa fragments, then the 100 kDa fragment was cleaved into the 70 kDa fragment. N-Terminal sequence analyses of PK-120 and its fragments demonstrated that it is a novel plasma protein, distinct from high molecular weight kininogen, a natural substrate for plasma kallikrein.

Effects of heparin on proteolytic activities in human plasma.

The influence of unfractionated heparin [average molecular weight (MW) 10,000-15,000 kD] and low-molecular-weight heparin (average MW 400-600 kD) on the plasma kallikrein-kinin and the fibrinolytic system was studied in vitro. Unfractionated heparin added to plasma gave an increase in kallikrein-like activity with a corresponding decrease of prekallikrein and functional kallikrein inhibition values. Plasmin-like activity was also increased, but minor changes in plasminogen and no change in antiplasmin values occurred. The changes were less pronounced with low molecular heparin compared with unfractionated heparin. The proteolytic changes were reversed by protamine sulfate but not influenced by the protease inhibitor aprotinin. Gel filtration yielded proteolytic activities able to split the plasma kallikrein substrate S-2305 and, to a minor degree, the plasmin substrate S-2251. The proteolytic activities were not due to complex formation with alpha 2-macroglobulin. We speculate that heparin binds to prekallikrein to form a heparin-prekallikrein complex which undergoes conformational changes and displays a kallikrein-like activity with the ability to split small synthetic substrates. Whether it is capable to split natural substrates remains unresolved.

Non-enzymic activation of the fifth component of human complement, by oxygen radicals. Some properties of the activation product, C5b-like C5

Purified human C5 was converted non-enzymically to an activated form as defined by its ability to participate in reactive lysis. This conversion occurred following exposure to systems that generate oxygen radicals, namely addition of H 2O 2 in the presence of ascorbic acid and iron or the addition of xanthine oxidase, acetaldehyde and iron. The conversion of C5 to a functionally active species was iron-dependent and inhibited by hydroxyl radical scavengers such as DMSO. The findings suggest that OH is the active oxygen species that converts C5. The conversion product of C5, termed C5(H 2O 2), is C5b-like due to its ability to bind C6 and cause reactive lysis. C5(H 2O 2) is much more stable than C5b obtained by complement convertases. Although C5(H 2O 2) has lost the binding site of native C5 for C3b it can be cleaved by complement-derived convertases; the cleavage is, however, less efficient than in the case of native C5. The resulting cleavage product, which is C5a-like, is chemotactic although C5(H 2O 2) is not chemotactic. C5(H 2O 2) serves as a better substrate for plasma kallikrein than native C5, resulting in the generation of a C5a-like chemotactic product. These data indicate that oxygen radicals can bring about a conformational change in C5, causing it to behave as a functionally activated molecule of the complement system. This may have implications for the role of complement and its activation in the inflammatory response.

Comparative study of two arginine ester hydrolases, E-I and E-II from the venom of Crotalus ruber ruber (red rattlesnake)

1. 1. Two arginine ester hydrolases, E-I and E-II from the venom of Crotalus ruber ruber were isolated and characterized. 2. 2. E-I and E-II have molecular weights of 32,000 and 33,000, and isoelectric points of 5.2 and 4.6, respectively. 3. 3. E-I and E-II are active upon the glandular kallikrein substrate, but neither enzyme was shown to have plasma kallikrein substrate hydrolytic activity. 4. 4. E-I has minimal fibrinogen-clotting activity. It was found to induce clotting by catalyzing the hydrolysis of only the A fibrinopeptide from the Aα-chain of fibrinogen.

Mechanism of kinin release from human low-molecular-mass-kininogen by the synergistic action of human plasma kallikrein and leukocyte elastase.

We have investigated the kinin release from human L-kininogen, a poor substrate for plasma kallikrein, by the synergistic action of human PMN elastase and plasma kallikrein. Although PMN elastase alone failed to generate kinin activity from L-kininogen, combination of PMN elastase with plasma kallikrein was found to be effective for the generation of kinin activity from L-kininogen. Two kinds of kinin, bradykinin and Met-Lys-bradykinin, were found to be released from L-kininogen by the synergistic action of PMN elastase and plasma kallikrein. Pretreatment of L-kininogen with PMN elastase facilitated the kinin release by plasma kallikrein, whereas pretreatment of L-kininogen with plasma kallikrein did not allow kinin release by the action of PMN elastase. These results suggested that PMN elastase would act firstly on L-kininogen to form a kinin containing fragment, from which kinin is released by the action of plasma kallikrein. The kinin-containing fragment was isolated by gel filtration and high-performance liquid chromatography of the elastase digest of L-kininogen. The amino-acid analysis and N-terminal amino-acid sequence analysis revealed that the kinin-containing fragment consisted of 26 amino-acid residues and is formed by cleavage of an Ile-Ser and a Ser-His bond of L-kininogen.

Assay for Hageman factor (factor XII) in human plasma by means of chromogenic substrate for plasma kallikrein

Determination of factor XII was performed in a test system where plasma samples were diluted (1/400-1/3200) in buffer and factor XII deficient plasma. Diluted Cephotest R was used as activator and after 10 min activation time chromogenic substrate H-D-Pro-Phe-Arg-pNA was added. The assay was adopted to an enzyme analyzer. The reproducibility of the method was acceptable with a coefficient of variation of 7.8% in the normal range. The correlation of this method to a one-stage clotting assay for factor XII was good, r 0.79. The preliminary reference interval was calculated to be 52–147% of normal (mean+ 2 S.D.).

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.

Demonstration of Kallikrein-Like Protease Activity in Nonactivated Plasma of Patients With Cooley's Anemia

Routine evaluation of 12 children with Cooley's anemia revealed that each one had a prolonged partial thromboplastin time. However, prothrombin time and thrombin time were within the normal range. Specific assays demonstrated low levels of the four contact factors: factors XI, XII, prekallikrein, and high molecular weight kininogen. Further investigation revealed activity against para-nitroanilide peptide substrates in unactivated plasma from all 12 patients. Following gel filtration on Sephadex G200, the activity emerged in one peak in the void volume, indicating a molecular weight of greater the 500,000. Activity was greatest against H-D-Pro-Phe-Arg-pNA, the substrate for plasma kallikrein, and was inhibited by diisopropyl fluorophosphate and trasolyl. It was unaffected by hirudin, soy bean trypsin inhibitor, and lima bean trypsin inhibitor. It was destroyed by heating at 56 degrees C. Specific antisera against human prekallikrein and human alpha-macroglobulin did not reduce the activity. It is concluded that a high molecular weight kallikrein-like protease, is present in the plasma of these patients. It is postulated that it is released into the circulation from tissue as a result of damage due to iron overload. It is further postulated that this protease brings about in vivo activation of the contrast factors, resulting in a fall in their circulating levels.

Demonstration of kallikrein-like protease activity in nonactivated plasma of patients with Cooley's anemia

Routine evaluation of 12 children with Cooley's anemia revealed that each one had a prolonged partial thromboplastin time. However, prothrombin time and thrombin time were within the normal range. Specific assays demonstrated low levels of the four contact factors: factors XI, XII, prekallikrein, and high molecular weight kininogen. Further investigation revealed activity against para-nitroanilide peptide substrates in unactivated plasma from all 12 patients. Following gel filtration on Sephadex G200, the activity emerged in one peak in the void volume, indicating a molecular weight of greater the 500,000. Activity was greatest against H-D-Pro-Phe-Arg-pNA, the substrate for plasma kallikrein, and was inhibited by diisopropyl fluorophosphate and trasolyl. It was unaffected by hirudin, soy bean trypsin inhibitor, and lima bean trypsin inhibitor. It was destroyed by heating at 56 degrees C. Specific antisera against human prekallikrein and human alpha-macroglobulin did not reduce the activity. It is concluded that a high molecular weight kallikrein-like protease, is present in the plasma of these patients. It is postulated that it is released into the circulation from tissue as a result of damage due to iron overload. It is further postulated that this protease brings about in vivo activation of the contrast factors, resulting in a fall in their circulating levels.

Determination of Prekallikrein in Plasma by Means of a Chromogenic Tripeptide Substrate for Plasma Kallikrein

Determination of Prekallikrein in Plasma by Means of a Chromogenic Tripeptide Substrate for Plasma Kallikrein

Determination of Prekallikrein in Plasma by Means of a Chromogenic Tripeptide Substrate for Plasma Kallikrein

A method for plasma prekallikrein determination utilizing a chromogenic tripeptide substrate is presented. The method has a good re-producibility and can easily be automized. Several parameters have been optimized. By using mixtures of deficient plasmas and pooled normal plasma or purified factors it was proved that prekallikrein was the factor determined and that more than 1O% (of normal plasma concentration) of FXII and HMW kininogen were essential for the activation of prekallikrein in our method. Further experiments showed that the method was fairly selective and was not influenced by inhibitors present in normal plasma. The later finding was attributed to the high dilution of plasma made possible by using a potent activator and a sensitive substrate.

783 PRESENCE OF A SERINE PROTEASE IN UNACTIVATED PLASMA OF CHILDREN WITH COOLEY'S ANEMIA

Biologic activity of factors X1, X11 and prekallikrein (PK) (chromogenic assay) was low in 10 patients aged 3-17 with Cooley's anemia; mean ± S.E., X1: 56%±6; X11: 51%±7; PK 59%±6. These levels were not due to generally impaired liver function as other clotting factors (1,11,V,V11,V111,1X & X) were in the normal range. The patients were studied to determine whether X1, X11 and PK were depressed due to 1) synthesis of biologically inactive factors or 2) in vivo factor activation followed by removal. Factor X11 measured by Laurell technique equalled biologic activity. Non-activated plasma cleaved the chromogenic substrate for thrombin S2238 (31 different samples from the 10 patients); mean nM p-nitroanaline (pNa) released/ml/min was 33±5 compared to 3±.4 in 20 plasma samples from 10 healthy controls (p < .001). Comparisons between 3 substrates showed greatest activity for 2302 (plasma kallikrein substrate) and least for 2222 (Xa) (mean nM pNa/ml/min: 2302: 185±27; 2238: 43±11, 2222: 11±4; p <0.001). Plasma activity was stable at -70° and was destroyed at 56°. It was not inhibited by the thrombin inhibitor Hirudin but was inhibited by DFP (serine protease inhibitor) and Trasylol (kallikrein inhibitor). This is the first demonstration of protease activity in unactivated plasma of patients who do not have DIC. We postulate that due to iron overload a zymogen (? prekallikrein) is activated to a protease (? kallikrein) which activates XI&XII with subsequent removal of XIa and XIIa.