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Recent evidence shows that many antiphospholipid antibodies (aPL) to negatively-charged phospholipid (PL) do not target anionic PL per se, but are specific for anionic PL-binding plasma proteins, for example, beta(2)-glycoprotein I (beta(2)-GPI) and prothrombin. We also reported that certain antiphosphatidylethanolamine antibodies (aPE) are not specific for phosphatidylethanolamine (PE) per se, but are directed to PE-binding plasma proteins, high molecular weight kininogen (HK), and low molecular weight kininogen (LK). Additional studies have shown that certain aPE failed to recognize purified kininogens but continued to produce aPE ELISA reactivity in the presence of semipurified HK preparations containing the HK binding proteins, factor XI (FXI) and prekallikrein (PK). We therefore investigated if certain of these aPE recognized FXI and/or PK. In this study we observed that aPE can recognize contact proteins FXI and PK independently or in combination with HK. Since contact proteins such as HK, PK and factor XII (FXII) have anti-coagulant and profibrinolytic functions, the pathophysiological role of aPE has yet to be elucidated. We propose that aPE of different specificities may initiate or promote characteristics pathological conditions in patients with thrombosis or recurrent pregnancy losses., (Copyright 2001 Academic Press.)

Kallikreins are serine proteases that catalyze the release of kinins and other vasoactive peptides. Previously, we have studied one tissue-specific (H. Yu et al., 1996, J. Am. Soc. Nephrol. 7: 2559-2564) and one plasma-specific (H. Yu et al., 1998, Hypertension 31: 906-911) human kallikrein gene in end-stage renal disease (ESRD). Short sequence repeat polymorphisms for the human plasma kallikrein gene (KLKB1; previously known as KLK3) on chromosome 4 were associated with ESRD in an African American study population. This study of KLKB1 in ESRD has been extended by determining the genomic structure of KLKB1 and searching for allelic variants that may be associated with ESRD. Exon-spanning PCR primer sets were identified by serial testing of primer pairs designed from KLKB1 cDNA sequence and DNA sequencing of PCR products. Like the rat plasma kallikrein gene and the closely related human factor XI gene, the human KLKB1 gene contains 15 exons and 14 introns. The longest intron, F, is almost 12 kb long. The total length of the gene is approximately 30 kb. Sequence of the 5'-proximal promoter region of KLKB1 was obtained by shotgun cloning of genomic fragments from a bacterial artificial clone containing the KLKB1 gene, followed by screening of the clones using exon 1-specific probes. Primers flanking the exons and 5'-proximal promoter region were used to screen for allelic variants in the genomic DNA from ESRD patients and controls using the single-strand conformation polymorphism technique. We identified 12 allelic variants in the 5'-proximal promoter and 7 exons. Of note were a common polymorphism (30% of the population) at position 521 of KLKB1 cDNA, which leads to the replacement of asparagine with a serine at position 124 in the heavy chain of the A2 domain of the protein. In addition, an A716C polymorphism in exon 7 resulting in the amino acid change H189P in the A3 domain of the heavy chain was observed in 5 patients belonging to 3 ESRD families. A third polymorphism in the coding sequence was a C699A shift that caused an amino acid change, H183Q. This allele was observed in 8 cases from 6 ESRD families but was not found in any control DNAs. Individually or combined, the allelic variants observed are not statistically associated with ESRD, though in several cases (e.g., H183Q) the small number of people in the population carrying these alleles limits our ability to statistically test for significant association with ESRD. Two new CA/GT repeat polymorphic markers, designated KLK3f and KLK3g, that have heterozygosities of 0.65 and 0.84, respectively, were identified within introns M and N. Analysis using the relative predispositional effect technique indicated that the frequencies of alleles 4 and 8 of KLK3f and allele 8 of KLK3g were significantly different between controls and ESRD cases. They accounted for 0.226, 0.096, and 0.313, respectively, in the probands of 166 ESRD families compared to 0.172, 0.066, and 0.244 in 139 healthy race-matched controls (allele P and total P < 0.05 for all three alleles). Therefore, although polymorphisms in the coding and 5'-proximal promoter of KLKB1 show no statistically significant association with ESRD in African Americans, there is still evidence for association of this part of chromosome 4 with ESRD. This observation suggests that other sequences within or near KLKB1, or another gene nearby, may contribute to ESRD susceptibility., (Copyright 2000 Academic Press.)

Background: Proteases and protease inhibitors are important in acute pancreatitis (AP), although little is known about the time course in cerulein-induced AP in the rat., Materials and Methods: AP was induced by supramaximal stimulation of cerulein, 10 microgram/kg/h, and during 72 h we measured lipase, amylase, albumin, prekallikrein, factor X, alpha(1)-protease inhibitor, alpha(1)-macroglobulin, alpha(2)-antiplasmin, antithrombin III (all in plasma) and macroscopic and histologic variables., Results: Within 12 h an edematous pancreatitis was evident with peak values of peritoneal exudate, pancreatic wet weight ratio, and plasma amylase and lipase activities. Histologically, edema and vacuolization were prominent already after 3 and 6 h, respectively, while inflammation, necrosis, and total histological score gradually increase to reach peak levels at 48 h. Proenzymes and most plasma protease inhibitors decreased to low levels after 6-12 h followed by a gradual increase. The sequential changes over time indicate that kallikrein - kinin activation, and plasminogen activation are probably early events in cerulein-induced AP in rats. alpha(1)-Macroglobulin and alpha(1)-protease inhibitor gradually decreased during the whole study period, probably being "second line" defense inhibitors. Levels above normal were seen for alpha(2)-antiplasmin and factor X at 48 h, normalizing at 72 h., Conclusions: These results suggest that protease activation and protease inhibitor consumption occur in cerulein-induced AP in the rat., (Copyright 1999 Academic Press.)

Beta Amyloid proteins (Abeta) of 38, 40, and 42 amino acids long were assessed for their ability to activate the plasma kinin-forming cascade in vitro. Incubation with a mixture of Factor XII (Hageman Factor), prekallikrein, and high-molecular-weight kininogen (HK) led to conversion of prekallikrein to kallikrein that was dependent on zinc ion. No activation occurred if Factor XII was omitted. There was rapid generation of bradykinin equal to the molar HK input indicating complete cleavage. Incubation of aggregated Abeta with diluted human plasma also led to prekallikrein activation and HK cleavage. Activation of the cascade by Abeta (1-38) was dependent upon its preincubation time in buffer, suggesting that aggregation of Abeta is required, and studies with Abeta (1-40) revealed time-dependent aggregation by microscopy and augmented zinc-dependent binding of both Factor XII and HK to aggregated Abeta. These data demonstrate that aggregated Abeta can bind and activate proenzymes of the plasma kinin-forming cascade in a zinc-dependent reaction to release bradykinin and is of sufficient potency to do so at physiologic concentrations of each protein and in the presence of naturally occurring protease inhibitors., (Copyright 1999 Academic Press.)

Incubation of heat-denatured plasma from the reticulated python, Python reticulatus with trypsin generated bradykinin-like immunoreactivity which was detected by radioimmunoassay using an antiserum raised against mammalian bradykinin. The primary structure of python bradykinin was established as Ala-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg. This amino acid sequence contains two substitutions (Arg1-->Ala and Ser6-->Thr) compared with mammalian bradykinin. Treatment of python plasma with glass beads under conditions previously shown to generate [Thr6]bradykinin in the plasma of a turtle and an alligator did not generate bradykinin-like immunoreactivity. The data indicate that the plasma of a lepidosaur contains a kininogen but, unlike the plasma of chelonians and crocodilians, does not contain a prekallikrein activator related to Factor XII.