The session on Contact Activation continued the discussion whether there is a correlation between decreased concentration of FXII and thromboembolism. In addition, since numerous new investigations have indicated that the kallikrein-kininogen system plays a major role in cellular as well as vascular development, we found it of great importance to focus also on these new observations which may be relevant to many unanswered clinical problems.

Venous thrombosis: Whether there is a correlation with venous thrombosis seems to depend upon the methodologic approach. A common denominator over the years is a frequency in normal individuals of 1-3 % having less then 50 % of FXII, while the frequency of FXII-deficiency among thrombotic patients is in the range of 7-10 %. An update from the Italian Registry of Congenital Coagulopathies indicates that the deficience of FXII measured by the classical APPT- test did not correlate convincingly with venous thromboembolism; however, this last study was prospective in nature and it seems to suggest that FXII- deficiency alone is not a risk factor for thrombosis.

A deficiency of FXII was reported to be caused by a genetic polymorphism (⁴⁶C/T in exon I of the 5 prime untranslated region) located close to the AUG translation codon of the FXII gene.

Also a significant number of thrombotic patients, when compared to normals, were deficient in either HK or prekallikrein.

The importance of FXII as a participant of an alternative pathway for plasminogen activation supports a correlation between Factor XII deficiency and thromboembolism. However, recent investigations have identified HK and PK as anti-thrombotic and the FXII and the HK/PK system is interrelated by activation. Both FXII and HK have been shown to be inhibitors of platelet activation.

Myocardial infarction: Studies have reported elevated levels of activated FXII (FXIIa) in patients with myocardial infarction. Although the method used in this study measured FXIIa, questions from the audience were related to the nature of the compound being measured. Nevertheless, this observation involves variations in concentration of a form of FXIIa circulating in plasma and may represent a risk factor for myocardial infarction. The levels of this nature of FXIIa appeared to increase further upon thrombolysis in patients with MI.

Regulation of cellular activity: The severity of FXII deficiency caused by the presence of auto antibodies seems to be far more serious than just the genetically determined deficiency. In a significant number of patients with Lupus- anticoagulants (LA) and primary anti-phospholipid antibody-syndrome (APS) the auto-antibody has been identified as anti-FXII antibody. Some of the features of these syndromes are thrombosis and fetal loss. While no correlation is found between antibodies to cardiolipin and/or ß₂-Glycoprotein I and recurrent fetal loss, a strong correlation was observed with the presence of anti-FXII antibodies. This suggests that FXII has a role in cellular development. Further studies are required to complement these observations, namely histology of the placentas from those individuals afflicted by recurrent fetal loss as well as the determination of cross- reactivity of these antibodies with plasminogen. It was noted that these antibodies do not cross-react with prothrombin.

In a controlled animal model of APS (NZW x BXSB F1 mice), auto-antibodies to HK were identified, providing an additional explanation for a thrombotic manifestation in APS. HK-knockout mice confirm that the kinin-kallikrein system is anti-thrombotic, pro-inflammatory and involved in angiogenesis. Anti-angiogenic properties of HK were Zn²⁺-dependent and limited to HKa and not to its single chain form in cell culture experiments. The inhibition was associated with apoptosis. The anti-angiogenic effect was mapped to the C-terminal region of the cell-binding domain, domain 5. The mechanisms behind these regulations remain to be determined. They may be several.

Additionally, a product of proteolytic cleavage of bradykinin by angiotensin converting enzyme (ACE) has been found to have anti-thrombotic properties. This proteolytic product corresponds to a pentapeptide, RPPGF.

Factor XI: Structure- function studies using recombinant proteins and monoclonal antibodies confirm regions within the molecule important for interacting with platelets, heparin, HK and FIX. Unlike previous mapping studies using synthetic peptides derived from different apple- domains, recombinant proteins revealed that apple II, and not apple I, is important for HK interaction; however, these findings did not rule out the possibility for apple I and apple II to participate in the interaction with HK and are thus in agreement with the data obtained using synthetic peptides. An interesting finding was that apple III is capable of binding both to platelets and FIX, thereby providing a reason for the dimeric nature of the FXI molecule. Finally, the FXIIa substrate binding region has not been identified yet by this last study.

The functional aspect of FXI as reflected by natural mutations and acquired inhibitors was discussed. It was noted that the mutation could be connected to either synthesis, secretion or activation. Several mutations affect Cys- Cys bonding. The mutation Cys ³⁵ => Arg in BHK transfected cells is associated with a lack of secretion. Another mutation not involving the Cys-Cys bonding, namely the Gly ⁵⁵⁵=> Glu, which is located within the catalytic domain, is associated with the presence of the antigen but with loss of function. The loss of function of this last mutation was determined by a combination assay where ultimately FXa activity was measured by a chromogenic substrate.

It was emphasized that all but one FXI- deficient patient had developed antibody inhibitors against FXI after transfusion. These antibodies were long-lasting. The majority of the antibodies (61.5 %) were associated with type II mutations. Thus, future studies aiming at a characterization of new mutations should start by evaluating the presence of type II mutations.

It was proposed that the molecular basis of platelet FXI-deficiency should be further analysed in view of the knowledge of unpublished data contradicting a result from a single published report.

Conclusions: The presentation of several animal models in this session indicates that the significance of proper plasma concentrations of FXII, HK and PK has an impact on the vascular system. It has been proposed that in order to correlate FXII, HK and PK- deficiencies with thromboembolism, the studies have to focus on individuals who have developed the disease as opposed to prospectively trying to identify these deficiencies as risk factors. The association with and activation of FXII and the HK/PK complex on the cell membranes, including the vascular wall, appear to have a hitherto unrecognised significance in cellular development, namely apoptosis, angiogenesis and possibly proliferation and differentiation. The newly observed properties of FXII and the kinin-kallikrein system indicates that these zymogens of proteolytic enzymes are involved in a variety of cardiovascular diseases and mutations in these proteins or activation disorders may result in serious pathological conditions related to the vascular system.

The Working Group to establish plasma standards for determination of FXII, HK, PK and FXI recommends that this work be considered a low priority task. This Working Group suggests high priority to establish a name other than "Contact Activation", which more precisely defines the function of FXII, XI, PK and HK in the vascular system.