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1. Monospecific antibodies against components of the guinea pig Hageman factor-dependent system, such as Hageman factor, prekallikrein and high-molecular-weight (HMW) kininogen were prepared. These antibodies demonstrated the presence of all of these components in the normal guinea pig skin extract with the properties identical to them in plasma on western immunoblotting analysis. 2. Guinea pig macroalbumin which is a major circulating inhibitor of activated Hageman factor was immunologically depleted from three animals. Duration of a vascular permeability reaction caused by an intradermal injection of activated Hageman factor was prolonged in all of these guinea pigs approximately twice as long as the control, indicating a presence of negative feedback regulation of the Hageman factor-dependent kinin generation cascade in dermal tissue via the circulating protease inhibitor.

We have shown that bovine HMW kininogen remarkably accelerates the activation of Factor XII and prekallikrein in the presence of kaolin, adsorbing on kaolin through the fragment 1.2 region and forming a complex with prekallikrein through the light chain region (Sugo et al., 1980; Ikari et al., 1981). The present study was undertaken to examine the role of HMW kininogen in the activation of Factor XII and prekallikrein with other negatively-charged surfaces. The activation system used here was as follows; (1) Activation of prekallikrein by Factor XII, (2) Activation of Factor XII by plasma kallikrein and (3) Activation of prekallikrein by Factor XIIa. Among a variety of foreign surfaces, amylose sulfate and sulfatide were the most efficient in the activation reaction of Factor XII and prekallikrein. Bovene HMW kininogen accelerated all the three reactions triggered by these surfaces. However, the accelerating effect of HMW kininogen on the activation of Factor XII by plasma kallikrein was very weak, when amylose sulfate or sulfatide was used as surface. The three reactions were highly dependent on the amounts of HMW kininogen and surfaces contained in the reaction mixtures. Excess amount of them inhibited these reactions. Among the various fragments, which were prepared from HMW kininogen digests with plasma and urinary kallikreins (Sugo et al., 1980), a large fragment consisting of fragment 1.2 and light chain accelerated the reactions. Thus both fragment 1.2 and the light chain region in HMW kininogen were essential for these activation reactions.

A zinc dependent serratial 56K protease caused enhancement of vascular permeability followed by edema formation when injected into the guinea pig peripheral cornea, the subconjunctival space, or the skin. Because this enhancement was not affected by antihistamine, involvement of the kinin-generating system in this permeability enhancement was investigated. The 56K protease induced permeability much greater extent than that by bradykinin on weight basis, and more so on molar basis. The phenomenon was inhibited by soybean trypsin inhibitor, a well known inhibitor of plasma kallikrein, and also by corn trypsin inhibitor, which is the best inhibitor of the activated Hageman factor. In vitro experiments using numbers of synthetic peptide substrates, the 56K protease exhibited a similar substrate specificity to that of plasma kallikrein. Kallikrein is a known endogenous activator of Hageman factor. The enhancement by 56K protease was greatly augmented by inhibition of kininase II with Glu-Trp-Arg-Pro-Gln-Ile-Pro-Pro-OH (SQ 20,881), suggesting generation of bradykinin. Thus, these results indicate that the enhancement of vascular permeability induced by the 56K protease is caused by an activation of Hageman factor by 56K protease followed by subsequent activation of cascade amplification, and resulted in kinin generation in vivo.

The possibility of an involvement of the kallikrein-kinin system in increasing vascular permeability induced by intradermal injection of the guinea pig activated Hageman factor (beta HFa) was examined. In vitro system, the kinin generation was observed in guinea pig plasma when the plasma was incubated with beta HFa. This kinin generation was dependent upon the dose of beta HFa added and upon the presence of plasma prekallikrein, since 97 percent of the kinin release was diminished by removing the prekallikrein in plasma by treatment with anti-prekallikrein antibody. These results suggested that the Hageman factor-kallikrein-kinin cascade in guinea pig was similar to those in human and bovine plasma. In the permeability experiment in vivo, a simultaneous injection of soybean trypsin inhibitor (10(-6) M), which is the inhibitor of guinea pig plasma kallikrein, inhibited the permeability response to beta HFa by more than 90 percent. The permeability response to beta HFa was attenuated 5 fold in animals depleted of the circulating plasma prekallikrein by intraarterial antibody administration. These results indicated the participation of plasma prekallikrein in the permeability reaction to beta HFa. A simultaneous injection of a kinin destructive enzyme, carboxypeptidase B, diminished the permeability reaction to beta HFa, without any inhibition of the amidolytic activity of beta HFa or plasma kallikrein. A simultaneous injection of an inhibitor of a kinin destructive enzyme (kininase II), SQ 20,881(Glu-Tyr-Pro-Arg-Pro-Gln-Ile-Pro-Pro-OH), augmented the permeability reaction to beta HFa 10 fold, without any effect on the amidolytic activity of beta HFa or plasma kallikrein.(ABSTRACT TRUNCATED AT 250 WORDS)

Previous studies from this department have established the presence of leukocyte migration inhibition activity in acute nonimmune inflammatory rat pleural exudates, induced by different irritants, which involved coagulation phenomenon. In the present investigation we present evidence for a role of kallikrein in the leukocyte migration inhibition phenomenon exhibited by acute pleural exudate in rates depleted of kininogens by intravenous injection of cellulose sulfate.

Studies of plasmas from individuals with Hageman trait (factor XII deficiency), plasma thromboplastin antecedent (PTA, factor XI) deficiency, Fletcher trait (plasma prekallikrein deficiency) and Fitzgerald trait (high molecular weight-kininogen deficiency) have revealed the importance of these proteins in blood coagulation. The interactions among them, however, are not fully elucidated. We have studied these reactions by two different approaches. (1) In a purified system, high molecular weight kininogen was absolutely required for activation of PTA by HF and ellagic acid (EA). The yield of activated PTA was proportional to the amount of HF, HMW-K, and PTA in the mixtures, suggesting that these three proteins may form a complex in the presence of EA. (2) In experiments with whole plasma, we took advantage of the adsorption of EA to Sephadex gels. When normal plasma or plasma deficient in HF, PK, HMW-K or PTA was exposed to Sephadex-EA and was separated by centrifugation, each supernatant plasma except that deficient in HF shortened the prolonged partial thromboplastin time (PTT) of HF-deficient plasma. Plasma simultaneously depleted of HMW-K, PK and PTA also shortened the PTT of HF-deficient plasma and of plasma depleted of HF and PK, but had virtually no procoagulant effect upon the PTT of plasma depleted of HF and MHW-K. Thus, exposure of HF in plasma to Sephadex-EA appeared to generate a clot-promoting form of HF in the absence of other clotting factors, but its expression required the presence of HMW-K.