Plasma Kallikrein-Kinin System

7 August 2005
8:30 to 12:00
Harbourside Meeting Room 3
Sydney Convention and Exhibition Centre

Chairman:  K. R. McCrae, USA
Co-Chairs:  R. A. DeLa Cadena, USA; D. Gailani, USA; M. J. Gallimore, UK; ; H. Saito, Japan; A. H. Schmaier, USA

The SSC on plasma kallikrein-kinin meeting was well attended with approximately 40 participants. The meeting was active, with much interesting discussion from most participants. Presentations covered a wide variety of topics ranging from standardization to reviews of basic science topics to clinical and human studies.

A brief review of the topics presented is described below:

Update on FXI Standardization (Dr. Elaine Gray)

Dr. Gray presented recent work focused on gaining acceptance of the WHO standard for FXI activity. At the same time, the SSC Lot 3 was assayed for FXI. This study involved 27 labs in a number of countries. The studies have been designed to compare the FXI levels in the Lot 3 standard with the current WHO international standard (IS) and “in-house” standards prepared in different labs participating in the study. Relevant results include the observation that the Lot 3 standard and IS are virtually identical in their FXI activity (0.88 vs 0.86 IU/ml) and that there is excellent interlaboratory agreement with these results. In comparison, the in-house standards showed significantly more variability. The potential sources of this variability was discussed, and may reflect ethnic differences in the local pools as well as other variables. A vote was taken as to whether the Lot 3 standard was felt to be acceptable for presentation to the ISTH for approval for submission to the WHO as consideration as an IS, and all attending were in favor. Endorsement was specifically provided by Dr. Gray, Dr. Keith McCrae, Dr. Robert Colman, Dr. Alvin Schmaier and Dr. Zia Shariat-Madar.

Prolycarboxypeptidase (PRCP): A new assay to assess PK activation on cells (Dr. Zia Shariat-Madar)

Dr. Shariat Madar reviewed work performed leading to the identication of PRCP as an endothelial cell PK activator, including the purification process leading to a 27019-fold purification that allowed identification of the enzyme. He also reviewed data demonstrating colocalization of PRCP with known HK receptors on endothelial cells, as well as functional data using PRCP antisense constructs. Future directions for this work, as well as possible ways to standardize PK activations assays were discussed by the group.

The intrinsic coagulation pathway is essential for arterial thrombus formation in mice (Dr. Thomas Renne’)

Dr. Renne discussed recently published studies from his own lab as well as that of Dr. David Gailani which have focused on coagulation and thrombosis in FXI and FXII deficient mice. Discussions included the importance of feedback activation of FXI by thrombin in FXI -/- mice, in which platelets adhere at the site of a wound but have deficient aggregation. Interestingly, FXII deficient mice were found to have no bleeding phenotype, but to have deficient thrombus formation in that occlusive thrombi did not form in these animals under circumstances in which they formed rapidly in wild-type mice. Possible reasons for this observation were discussed at length by the group. The use of FXII inhibitors as thromboprotectants was also considered, and thought to be worthy of future consideration.

Update on development of thrombostatin (Dr. Alvin Schmaier)

Dr. Schmaier discussed thrombostatin, the RPPGF product derived from bradykinin. This agent has been shown to effectively inhibit coagulation in mice, including thrombin induced platelet aggregation. Studies that have led to further identificagion of thrombostatin mechanisms were reviewed, including its ability to bind to peptidic thrombin cleavage sites of PARs (1 and 4) and inhibit PAR cleavage. Role of specific amino acids in these interactions were defined, in particular a specific arginine residue and proline 46 of PAR 4. Finally, the development of new thrombostatin peptidomimetics, including FM19, was reviewed. The potential clinical utility of these agents was discussed at length by the group.

Kininogen in inflammatory bowel disease and reactive arthritis (Dr. Robert Colman)

Dr. Colman reviewed the pathogenesis of IBD and arthritis and the animal models used to study these disorders. The broad range of activities of BK and HK in the pathogenesis of inflammatory disorders was described in detail. The marked efficacy of a kallikrein inhibitor P8780 in preventing IBD and the systemic symptoms of inflammation which accompany gut disease was presented and discussed by the group, and the reasons for differences in disease susceptibility between Lewis and Buffalo-Fisher rats, which relate to the susceptibility of HK cleavage due to an HK polymorphism between these strains was described. Dr. Colman also described the development of an HK deficient rat (and wild type control) by back crossing of B/N Katholiek HK deficient rats with common rat strains was presented. Studies with these animals demonstrated an important role for HK in the development of inflammatory disease.

Kininogen and angiogenesis: A review (Dr. Keith McCrae)

Dr. McCrae discussed several areas relating to the role of HK/HKa in regulation of angiogenesis. The antiangiogenic activity of HKa and HK domain 5 was reviewed, as well as the current mechanisms that have been proposed to account for this activity. Studies focused on means by which the active domain 5 structure may be defined were discussed. Characterization of the murine kininogen gene locus was also reviewed, including the recent finding that two kininogen genes occur in the mouse and rat. The development of a mouse deficient in the murine kininogen gene 1 was discussed, as well as preliminary data suggesting that this gene, KNG1, is the source of plasma kininogen.

ELISAs for FXIIa and kallikrein-C1INH complexes: preliminary data from the second NorthwickPark Heart Study (Dr. Jose’ W.P. Govers-Riemslag)

Dr. Govers-Riemslag discussed several aspects of the measurement of these complexes in patients enrolled in the NPHS, who were healthy men aged 52-61 without known heart disease. Extensive detail concerning the development and standardization of these assays was provided, demonstrating linearity of log-transformed plots using standards developed specifically for this purpose. The study was a nested case control study involving 829 men, 231 with CHD, 56 with stroke). Many results were presented, but among these one of the most important was that the highest amounts of CHD occurred in patients in whom the levels of FXIIa:C1INH levels were in the lowest tertile. There was not a strong correlation between these levels and free FXIIa. The relationship of these findings to the findings of Dr. Renne’ in FXII deficient mice was discussed by the group.

Detection of activated FXIIa in humans (Dr. David Pritchard)

Dr. Pritchard discussed the use of a specific monoclonal antibody that recognizes FXIIa with >10 8 fold sensitivity greater than FXII to study plasma FXIIa. Most of FXIIa is found in the fluid phase of blood, with some distributed to lipids and cells. Interestingly, significant amounts of FXIIa occurred on cells even in FXII deficient patients. Other molecules with which FXIIa associated included ApoA1 and E, C1INH, kallikrein and kininogen. The fact that the 78 kDa form of FXIIa increases after MI/thrombolysis was discussed, and the potential role of thrombolysis per se in inducing these elevations was discussed.

At the conclusion of the meeting, Dr. McCrae thanked all participants, and encouraged communication of new ideas and proposals for more discussions at future SSC meetings. A major goal of this subgroup is to expand and make others aware of the broad reaching physiologic importance of the kallikrein-kinin system.

Submitted 8/9/05

Keith R. McCrae, M.D.