Fitzgerald Trait Research Articles

Plasma kallikrein: Novel functions for an old protease

Editorial on Bird et al. Thromb Haemost 2012; 107: 1141-1150. Plasma kallikrein (PK) is a plasma protease that drives procoagulantand proinflammatory reactions via the intrinsic pathway of coagulation and the kallikrein-kinin system, respectively. The structure and biochemistry of PK have been analysed in great detail; however, its in vivo functions are just beginning to emerge. In this issue of Thrombosis and Haemostasis, Bird et al. presents a mouse model with target ablation of the PK gene (1). Severe deficiency of PK abolishes experimental thrombosis in arterial and venous vascular beds. Despite the striking thromboprotective effects conferred by targeting the protease, PK deficiency has minor effect on the haemo static capacity of gene-deficient mice. This animal model provides a promising tool to study the pathogenetic role of the protease in models of thrombosis and inflammation with exciting therapeutic perspectives. In the original waterfall cascade model of plasma coagulation, fibrin formation may be initiated by two distinct pathways, triggered either by exposure of blood to tissue factor at an injured vessel wall or to blood-borne (intrinsic) factors. The intrinsic pathway of coagulation is initiated by factor (F)XII, in a reaction involving highmolecular-weight kininogen (HK) and PK, collectively referred to as the plasma contact system (2). Contact with negatively charged surfaces such as kaolin or polyphosphate activates FXII, and activated FXII (FXIIa) cleaves PK to generate active kallikrein, which in turn reciprocally activates FXII zymogen. FXIIa initiates fibrin formation through its principal substrate of the intrinsic coagulation pathway, FXI and also triggers liberation of the potent inflammatory mediator bradykinin (BK) by PK-mediated HK cleavage (3). Binding of BK to the kinin B2 receptor (B2R) causes an immediate nitric oxide-mediated vasodilation through an increase of cGMP in vascular smooth muscle cells, activates various pro-inflammatory signalling pathways that dilate vessels, induces chemotaxis of neutrophils and increases vascular permeability and fluid efflux (4). In a positive feedback loop, kallikrein also activates surfacebound FXII to FXIIa. Furthermore, PK has the capacity to convert pro-urokinase to urokinase, plasminogen to plasmin, and for triggering the renin-angiotensin system. Due to its BK-forming capacity, kallikrein has potent in vitro effects on leukocyte migration, stimulates monocytes, and induces neutrophil aggregation. Recent studies have suggested a role of PK for control of intracerebral haemorrhage in hyperglycaemia (5). In contrast, hereditary deficiencies in PK (Fletcher trait) and its substrate HK (Fitzgerald trait) were not associated with haemostatic abnormalities (6). The serpin C1 esterase inhibitor (C1INH) is the major plasma inhibitor of PK and controls enzymatic activity of the protease. Plasma prokallikrein (PPK) is the zymogen of the trypsin-like serine protease PK. PPK is expressed by hepatocytes in humans and mice and circulates in plasma at a concentration of 50 g/ml, predominantly in a noncovalent 1:1-complex with HK. PK is a paralog of FXI, and both genes have evolved from a common anchestor (7). The principal mechanism of PK is to cleave the high-molecular-weight precursor (HK) that liberates BK. Despite similar names, PK is not related to the large group of kininogenases called tissue-type (urinary or glandular) kallikreins. Analogous to FXI, PK is composed of four apple domains (Apple 1–4) that form the heavy chain of the molecule and a C-terminal protease domain (8). However, in contrast to human FXI, human PK is a monomer. PK migrates as a doublet with 86and 88-kDa on nonreducing SDS-polyacrylamide gels. The two bands represent differentially glycosylated proteins. Contact activation is the mechanism of contact-induced FXII zymogen activation. Contact activation provides the mechanistic basis for one of the most commonly used diagnostic clotting test the activated partial thromboplastin time (aPTT). The contact surface used in aPTT assays is typically a negatively charged non-physiological substance such as kaolin (aluminum silicate), celite (diatomaceous earth), or ellagic acid. The aPTT is widely used in clinical practice for preoperative screening and monitoring of anticoagulant therapy. Despite its important role in fibrin formation in the aPTT assay, PK-driven coagulation does not appear to have haemo static or other physiological functions in vivo. This notion is based on the observation that PK deficiency, a rare incidentally observed coagulation abnormality in humans, has not yet been associated with pathology, such as abnormal bleeding, despite causing a marked prolongation of the aPTT. Similar to PK-deficient individuals, humans lacking the other contact proteins, FXII or HK, do not have impaired haemo stasis. Recently, FXII-deficient (FXII-/-) mice were generated and phenotyped to study the function of coagulation FXII in vivo (9, 10). Similar to FXII-deficient humans, FXII-/mice have a normal haemostatic capacity as assessed by a tail-bleeding assay. Completely unexpected, intravital fluorescence microscopy and blood flow measurements in three distinct arterial beds revealed a severe defect in FXII-defi-

Studies on Fletcher trait and Fitzgerald trait

The way by which contact of blood with foreign surface accelerates clotting has been elucidated from the discovery of four rare disorders of blood coagulation; Hageman trait, plasma thromboplastin antecedent (PTA) deficiency, Fletcher trait, and Fitzgerald trait. Interestingly, it was unexpectedly found that Fletcher factor is plasma prekallikrein and Fitzgerald factor is high-molecular-weight kininogen; components of the kinin-generating system, thus disclosing intimate relationships among clotting, fibrinolysis and kinin generation which may be viewed as body's defense reactions against injury. This review mainly reflects our research on Fletcher trait and Fitzgerald trait during the 1970s in Cleveland.

Characterization of molecular defects of Fitzgerald trait and another novel high-molecular-weight kininogen-deficient patient: insights into structural requirements for kininogen expression

AbstractA 6-year-old male with vertebral-basilar artery thrombosis was recognized to have high-molecular-weight kininogen (HK) deficiency. The propositus had no HK procoagulant activity and antigen (< 1%). Using monoclonal antibodies (Mabs) to kininogen domain 3, the propositus, family members, and Fitzgerald plasma were determined to have detectable low-molecular-weight kininogen. Mabs to HK domains 5 and 6 do not detect HK antigen in the propositus’ plasma. The propositus has a single base pair (bp) deletion in cDNA position 1492 of exon 10 affecting amino acid 480 of the mature protein and resulting in a frameshift and a premature stop codon at position 1597 (amino acid 532). Unexpectedly, Mabs to the heavy chain and domain 5 of HK detect a 92-kDa form of HK in Fitzgerald plasma, the first HK-deficient plasma. The 92-kDa Fitzgerald HK has amino acid residues through 502, corresponding to domains 1 through 5, but lacks epitopes of domain 6 (positions 543 to 595). Fitzgerald DNA has a normal exon 10, but a 17-bp mutation in intron 9. These combined results indicate that mutations in the kininogen gene may differentially affect biosynthesis, processing, and/or secretion of HK.

Rapid Direct Determination of Low and High Molecular Weight Kininogen in Human Plasma by Particle Concentration Fluorescence Immunoassay (PCFIA)

In this study, we employed Particle Concentration Fluorescence Immunoassay (PCFIA), for directly measuring both high and low molecular weight kininogens (HK and LK) in human plasma. In 38 normal donors, the mean values for plasma kininogens were 93 mg/ml +/- 19 SD, 82 mg/ml +/- 12 SD, and 175 mg/ml +/- 29 SD, respectively for LK, HK, and TotK (the sum of LK and HK detected by their common heavy chains). Plasma completely deficient in HK and LK was unreactive (< 0.25 mg/ml) in all 3 assays whereas plasma from a patient with Fitzgerald Trait had an HK value of 11 mg/ml, an LK value of 36 mg/ml and a TotK value of 59 mg/ml. The reagents can be prepared in advance and all three kininogen determinations can be performed, using the same diluted sample on 24 plasma samples, in triplicate, or 40 plasma samples, in duplicate, in less than 1 h. By performing all 3 kininogen determinations, it is possible to differentiate cleaved from intact kininogens. This technique will facilitate the widespread screening of kininogen levels in biological fluids of normal humans as well as of patients with various diseases.

Anticoagulant Activity in Cell Homogenate of Adult T Cell Leukemia

The hemostatic abnormality in 18 patients with adult T cell leukemia (ATL) was studied. Activated partial thromboplastin time (APTT) was slightly prolonged and prekallikrein activity was markedly low in these patients. The leukemic cell homogenate from these patients prolonged the recalcification time (RCT) of normal plasma; homogenates containing more than 3 x 10(3) cells/microliter prolonged it, although a lower cell concentration shortened it. The crude anticoagulant fraction from the gel filtration, with a molecular weight of about 34,000, prolonged RCT. The crude anticoagulant did not affect prothrombin time (PT), thrombin activity or activated X activity at any concentration, but prolonged the contact activation test, inhibited the activation of prekallikrein and prolonged RCT of Fletcher trait, Fitzgerald trait and F XII deficient plasma. These effects of ATL cell homogenate were stronger on platelet poor plasma than on platelet rich plasma. Although ATL cells had low procoagulant activity, increase of leukemic cells made anticoagulant activity predominant, might be the cause of hemostatic abnormality or amplify the bleeding tendency in patients with ATL.

Characterization of guinea-pig high-molecular-weight kininogen as multi-functional molecule

High-molecular-weight (HMW) kininogen was purified from guinea-pig plasma by measuring its ability to correct the prolonged clotting time in human HMW kininogen deficient plasma (Fitzgerald trait). The purified HMW kininogen demonstrated a homogeneous band in disc gel electrophoresis in the presence of sodium dodecyl sulfate under reducing or non-reducing conditions with an apparent molecular weight of 100 000. Kinin released from HMW kininogen by treatment with guinea-pig plasma kallikrein was identified as bradykinin by reverse-phase HPLC and amino-acid analysis. The capacity of HMW kininogen as a thiol-proteinase inhibitor was realized by its dose-dependent inhibitory activity to papain. The K i value for papain was estimated to be 42 pM. The kinin-free HMW kininogen maintained the inhibitor and clotting-factor activities with similar capacities to those of the HMW kininogen molecule. Heavy chain (H-chain) and light chain (L-chain) of HMW kininogen were prepared from reduced and alkylated kinin-free HMW kininogen by HPLC. The S-alkylated H-chain, but not L-chain, demonstrated the inhibitor activity with the K i value 6.9 nM for papain, whereas the S-alkylated L-chain, but not H-chain, maintained the clotting activity one-third of the capacity of HMW kininogen. Specific antibodies recognized HMW kininogen, but also a probable low-molecular-weight kininogen(s) with an apparent molecular weight of 60 000 in the guinea-pig plasma. All of these properties are consistent with the reports on human, bovine and rat HMW kininogen.

Rat plasma high-molecular-weight kininogen. A simple method for purification and its characterization.

High-molecular-weight kininogen has been isolated from rat plasma in three steps in a relatively high yield. The purified preparation gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence and presence of 2-mercaptoethanol, and the apparent Mr was estimated as 100,000. On incubation with rat plasma kallikrein, rat high Mr kininogen yielded a kinin-free protein consisting of a heavy chain (Mr = 64,000) and a light chain (Mr = 46,000), liberating bradykinin. The kinin-free protein was S-alkylated, and its heavy and light chains were separated by a zinc-chelating Sepharose 6B column. The amino acid compositions of rat high Mr kininogen and its heavy and light chains were very similar to those of bovine high Mr kininogen and its heavy and fragment 1.2-light chains, respectively. A high histidine content in the light chain of rat high Mr kininogen indicated the presence of a histidine-rich region in this protein as in bovine high Mr kininogen, although this region was not cleaved by rat plasma kallikrein. Rat high Mr kininogen corrected to normal values the prolonged activated partial thromboplastin time of Brown-Norway Katholiek rat plasma known to be deficient in high Mr kininogen and of Fitzgerald trait plasma. The kinin-free protein had the same correcting activity as intact high Mr kininogen. Rat high Mr kininogen also accelerated approximately 10-fold the surface-dependent activation of rat factor XII and prekallikrein, which was mediated with kaolin, amylose sulfate, and sulfatide. These results indicate that rat high Mr kininogen is quite similar to human and bovine high Mr kininogens in terms of biochemical and functional properties.

Prolonged activated partial thromboplastin time and deficiency of high molecular weight kininogen in brown norway rat mutant (Katholiek strain)

Activated partial thromboplastin time (APTT) was examined in Brown Norway (B/N) Katholiek rat, which was previously reported as high molecular weight kininogen deficient. APTT of B/N Katholiek was prolonged to 35 sec in comparison with B/N Kitasato and SD rat, showing APTT of 22–24 sec. The mixture of B/N Katholiek plasma and B/N Kitasato plasma (1:1) showed normal APTT value. B/N Katholiek plasma corrected the abnormally prolonged human coagulation factor deficient plasmas, such as XI, XII and prekallikrein deficient plasmas, while it did not correct the APTT of HMW kininogen deficient, Fitzgerald plasma. Intravenous injection of bromelain, which was previously reported to produce prolonged hypotension through the activation of factor XII to release bradykinin, induced slight effect in Katholiek rat, while in Kitasato rat it showed prolonged hypotension in similar degree as SD rat. Contents of coagulation factors in B/N Katholiek thus measured as well as the values of prekallikrein and HMW kininogen previously reported were summarized and suggested that B/N Katholiek rat could be similar deficiency as Fitzgerald trait.

Factor XI Adsorption to Surface: Interaction of High Molecular Weight Kininogen (HMWK) and a Plasma Adsorption Inhibitor

High molecular weight kininogen (HMWK) plays an important role in surface activation of factor XI. These experiments were undertaken to evaluate the role of HMWK in factor XI adsorption to glass using purified 125I-labeled factor XI. Factor XI binding from Fitzgerald trait plasma was markedly reduced compared to normal plasma or other plasmas deficient in contact phase clotting factors; addition of exogenous HMWK to normal plasma levels corrected this defect. However, addition of higher concentrations of HMWK reduced factor XI binding. Addition of HMWK to normal plasma also reduced factor XI binding. In purified solutions, HMWK reduced factor XI binding to glass. Exposure of the glass to HMWK prior to addition of factor XI had no effect on factor XI binding. Factor XI adsorption from all plasmas was much less than from citrate-saline, and factor XI binding from both normal and Fitzgerald trait plasmas increased with increasing dilutions of the plasmas. These results lead to the conclusion that the direct effect of HMWK on factor XI is to reduce its ability to bind to glass by formation of a less adsorbable complex. The effect observed in Fitzgerald trait plasma, of increased factor XI binding on initial addition of kininogen, is an indirect effect achieved by counteracting another normal plasma activity which inhibits factor XI adsorption.

Relationship between structure and correcting activity of bovine high molecular weight kininogen upon the clotting time of fitzgerald-trait plasma

Bovine high molecular weight kininogen (bHMWK) partially corrects the activated plasma thromboplastin time (aPTT) of Fitzgerald trait plasma which is congenitally deficient in HMWK. The relationship between the structure and activity of HMWK was clarified by studying the effects of different fragments of bHMWK on the aPTT of Fitzgerald-trait plasma. The peptides studied were lys-bradykinin-free HMWK, bradykinin-fragment 1-2-free HMWK, heavy chain, fragment 1-2-light chain, and light chain. All fragments were tested in equimolar concentrations. Bradykinin-fragment 1-2-free HMWK, heavy chain, and light chain have little or no correcting activity upon Fitzgerald-trait plasma aPTr. Fragment 1-2 light chain has the same correcting activity as intact bHMWK, while that of lys-bradykinin-free HMWK appears to be higher. Both fragment 1-2 and fragment 2 inhibit the clotting time of normal human plasma. When compared on a molar basis, fragment 2 is a more active inhibitor than fragment 1-2. When the effects of bovine plasma kallikrein upon bHMWK and hHMWK were studied, it was found that it released kinins from both kininogens. However, while the correcting activity of bHMWK was completely destroyed after 60 min of incubation, that of hHMWK was fully retained. These data suggest that: (a) the active part of bHMWK is comprised of the fragment 1-2 light chain portion; (b) fragment 1-2 or fragment 2 is the binding site to negatively charged surfaces, while the light chain interacts with other components of the surface-mediated reactions; and (c) bovine plasma kallikrein releases kinins, but probably does not cause the release of fragment 1-2 from human HMWK.

Activated Partial Thromboplastin Time and Minor Coagulopathies

Five commercially available activated partial thromboplastin time (APTT) test systems were compared with the kaolin partial thromboplastin time (KPTT) method to determine sensitivity in detecting minor coagulation defects. All reagent systems detected severe factor VIII-, IX-, and XI-deficient hemophilia. Homozygous states of factor XII deficiency, Fletcher factor deficiency, and high-molecular-weight kininogen deficiency (Fitzgerald trait) also showed abnormally long APTTs by all systems. Of 19 samples from patients with deficiencies of factors XII, VIII, IX, XI, and II ranging from 2.5 to 52%, eight had deficiencies that were not detected by reagent A (ellagic acid); two, by reagent B (ellagic acid); two, by reagent C (kaolin); one, by reagent D (silica); one, by the KPTT method. All deficiencies were detected by reagent E (celite). Heparin effect on plasma was less well detected by reagent A (ellagic acid) than with the other test systems. APTT test systems can vary greatly in their abilities to detect minor coagulation abnormalities.

Plasma thromboplastin antecedent (PTA, factor XI): a specific and sensitive radioimmunoassay

A specific, sensitive, and reproducible radioimmunoassay for human plasma thromboplastin antecedent (PTA, factor XI) has been developed with purified PTA and monospecific rabbit antiserum. Precise measurements of PTA antigen were possible for concentrations as low as 0.3% of that in normal pooled plasma. Normal plasma contained approximately 6 microgram PTA/ml. A good correlation (correlation coefficient 0.68) existed between the PTA procoagulant assays and radioimmunoassays among 50 normal adults (25 males and 25 females). PTA antigen was markedly reduced in plasma of 13 patients with congenital homozygous PTA deficiency (range less than 0.003–0.128 U/ml) and 9 patients with hepatic cirrhosis (0.35+/-0.17 U/ml), but was normal in those of 9 patients under treatment with warfarin, 8 patients with disseminated intravascular coagulation and 16 patients with other congenital clotting factor abnormalities, including prekallikrein deficiency (Fletcher trait) and high molecular weight kininogen deficiency (Fitzgerald trait).

Plasma Thromboplastin Antecedent (PTA, Factor XI): A Specific and Sensitive Radioimmunoassay

A specific, sensitive, and reproducible radioimmunoassay for human plasma thromboplastin antecedent (PTA, factor XI) has been developed with purified PTA and monospecific rabbit antiserum. Precise measurements of PTA antigen were possible for concentrations as low as 0.3% of that in normal pooled plasma. Normal plasma contained approximately 6 microgram PTA/ml. A good correlation (correlation coefficient 0.68) existed between the PTA procoagulant assays and radioimmunoassays among 50 normal adults (25 males and 25 females). PTA antigen was markedly reduced in plasma of 13 patients with congenital homozygous PTA deficiency (range less than 0.003-0.128 U/ml) and 9 patients with hepatic cirrhosis (0.35+/-0.17 U/ml), but was normal in those of 9 patients under treatment with warfarin, 8 patients with disseminated intravascular coagulation and 16 patients with other congenital clotting factor abnormalities, including prekallikrein deficiency (Fletcher trait) and high molecular weight kininogen deficiency (Fitzgerald trait).

Purification of high molecular weight kininogen and the role of this agent in blood coagulation.

Recent studies of individuals with high molecular weight (HMW) kininogen deficiency established the importance of this plasma protein for in vitro initiation of blood coagulation. In the present study, HMW-kininogen was highly purified from human plasma by monitoring its clot-promoting activity, using Fitzgerald trait plasma as a substrate. This preparation of HMW-kininogen revealed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (mol wt: 120,000) and released 1% of its weight as bradykinin upon incubation with plasma kallikrein. HMW-kininogen specifically repaired impaired surface-mediated plasma reactions of Fitzgerald trait plasma, but did not affect those of Hageman trait and Fletcher trait plasma. Kinin release from HMW-kininogen by trypsin, but not by plasma kallikrein, resulted in total loss of clot-promoting activity. No inhibitors of coagulation were found when all kinin activity was removed from HMW-kininogen by trypsin. The roles of HMW-kininogen, Hageman factor (HF, Factor XII), plasma prekallikrein (Fletcher factor), and plasma thromboplastin antecedent (PTA, Factor XI) in blood coagulation were studied in a purified system. HMW-kininogen was absolutely required for activation of PTA by HF and ellagic acid. The yield of activated PTA was proportional to the amount of HF, HMW-kininogen, and PTA in the mixtures, suggesting that, to activate PTA, these three proteins might form a complex in the presence of ellagic acid. No fragmentation of HF was found under these conditions. In contrast to HF, HF-fragments (mol wt: 30,000) activated PTA in the absence of HMW-kininogen and ellagic acid. Thus, it appears that in the present study PTA was activated in two distinct ways. Which pathway is the major one in whole plasma remains to be determined.

Prekallikrein deficiency in a kindred with kininogen deficiency and Fitzgerald trait clotting defect. Evidence that high molecular weight kininogen and prekallikrein exist as a complex in normal human plasma.

Plasma from an individual with a hereditary deficiency of kininogens is deficient in kininogen antigens; heterozygous relatives are partially deficient in plasma kininogen antigens. In addition, plasma from the proband is partially deficient in functional and antigenic properties of a plasma prekallikrein, and the relatives heterozygous for kininogen deficiency are also partially deficient in the plasma prekallikrein. It is possible that the defects are both inherited and that the inheritance of a deficiency of prekallikrein is genetically linked to the inheritance of a deficiency of kininogen. Alternatively, it is possible that the deficiency of prekallikrein may be due to its hypercatabolism which could be a consequence of a deficiency of high molecular weight kininogen that may stabilize the prekallikrein in plasma. Evidence to support this possibility is presented by the fact that prekallikrein and high molecular weight kininogen apparently exist as a complex in normal plasma, because monospecific antiserum to kininogen removed both high molecular weight kininogen and prekallikrein from plasma, and vice versa. Moreover, prekallikrein was not adsorbed from kininogen-deficient plasma by antiserum to kininogen unless high molecular weight kininogen was first added to the plasma. Low molecular weight kininogen did not participate in these reactions.

Proactivator and activator levels of plasminogen in plasma as measured by caseinolysis.

A procedure is presented for the caseinolytic assay of plasminogen proactivator and activator levels in blood. The validity of the method was established and normal plasma proactivator levels were determined in 10 subjects. No detectible activator activity was found in these subjects. Venous occulsion produced a small rise in activator activity. Hageman-deficient and Fitzgerald trait plasmas did not produce activator activity after exposure to kaolin. The generation of activator activity in the Fitzgerald plasma was partly restored by the addition of purified high molecular weight kininogen, but the Hageman-deficient plasma was not corrected. The caseinolytic assay avoids some of the drawbacks which are inherent in methods which depend on fibrin dissolution for the detection of enhanced fibrinolytic states, provides a reproducible baseline, and may be sufficiently sensitive to detect changes of clinical interest.

Fitzgerald factor (high molecular weight kininogen) clotting activity in human plasma in health and disease in various animal plasmas

Fitzgerald factor (high molecular weight kininogen) is an agent in normal human plasma that corrects the impaired in vitro surface- mediated plasma reactions of blood coagulation, fibrinolysis, and kinin generation observed in Fitzgerald trait plasma. To assess the possible pathophysiologic role of Fitzgerald factor, its titer was measured by a functional clot-promoting assay. Mean +/- SD in 42 normal adults was 0.99+/-0.25 units/ml, one unit being the activity in 1 ml of normal pooled plasma. No difference in titer was noted between normal men and women, during pregnancy, or after physical exercise. Fitzgerald factor activity was significantly reduced in the plasmas of eight patients with advanced hepatic cirrhosis (0.40+/-0.09 units/ml) and of ten patients with disseminated intravascular coagulation (0.60+/-0.30 units/ml), but was normal in plasmas of patients with other congenital clotting factor deficiencies, nephrotic syndrome, rheumatoid arthritis, systemic lupus erythematosus, or sarcoidosis, or under treatment with warfarin. The plasmas of 21 mammalian species tested appeared to contain Fitzgerald factor activity, but those of two avian, two repitilian, and one amphibian species did not correct the coagulant defect in Fitzgerald trait plasmas.

Fitzgerald Factor (High Molecular Weight Kininogen) Clotting Activity in Human Plasma in Health and Disease in Various Animal Plasmas

AbstractFitzgerald factor (high molecular weight kininogen) is an agent in normal human plasma that corrects the impaired in vitro surface-mediated plasma reactions of blood coagulation, fibrinolysis, and kinin generation observed in Fitzgerald trait plasma. To assess the possible pathophysiologic role of Fitzgerald factor, its titer was measured by a functional clot-promoting assay. Mean ± SD in 42 normal adults was 0.99 ± 0.25 units/ml, one unit being the activity in 1 ml of normal pooled plasma. No difference in titer was noted between normal men and women, during pregnancy, or after physical exercise. Fitzgerald factor activity was significantly reduced in the plasmas of eight patients with advanced hepatic cirrhosis (0.40 ± 0.09 units/ml) and of ten patients with disseminated intravascular coagulation (0.60 ± 0.30 units/ml), but was normal in plasmas of patients with other congenital clotting factor deficiencies, nephrotic syndrome, rheumatoid arthritis, systemic lupus erythematosus, or sarcoidosis, or under treatment with warfarin. The plasmas of 21 mammalian species tested appeared to contain Fitzgerald factor activity, but those of two avian, two reptilian, and one amphibian species did not correct the coagulant defect in Fitzgerald trait plasmas.

Effect of bovine high molecular weight kininogen and its fragments on fitzgerald trait plasma

Fitzgerald trait, a deficiency of high molecular weight (HMW) kininogen, is characterized by a prolongation of both the activated partial thromboplastin time (aPTT) and of the kaolin-activated euglobulin lysis time. To study the role of HMW kininogen in coagulation and fibrinolysis, highly purified bovine HMW kininogen, kinin-free HMW kininogen, a histidine-rich fragment of HMW kininogen, and low molecular weight (LMW) kininogen were obtained. The effects of these proteins on the aPTT and kaolin-activated euglobulin lysis time of Fitzgerald trait plasma were studied. It was found that whole bovine plasma and HMW kininogen had identical capacities to partially correct the prolonged aPTT. Kinin-free HMW kininogen had only 30% of the correcting capacity of intact HMW kininogen. LMW kininogen had no effect on the aPTT of Fitzgerald plasma. The histidine-rich fragment prolonged the aPTT of Fitzgerald plasma and of normal human plasma without affecting the one-stage prothrombin time. HMW kininogen also corrected the kaolin-activated euglobulin lysis time of Fitzgerald plasma. Kinin-free HMW kininogen and LMW kininogen had weak capacities to correct the Fitzgerald kaolin-activated euglobulin lysis time. The histidine-rich fragment significantly prolonged the kaolin-activated euglobulin lysis time of Fitzgerald and normal plasmas. The histidine-rich fragment appears to inhibit the activation of Hageman factor (Factor XII) by kaolin or ellagic acid. Our data suggest that intact bovine HMW kininogen corrects the deficiency of Fitzgerald trait plasma; but the hydrolysis of HMW kininogen by plasma kallikrein yields peptides with decreased correcting activity, and at least one with inhibitory activity in contact-mediated coagulation and fibrinolysis.

Evidence that Fitzgerald factor counteracts inhibition by kaolin or ellagic acid of the amidolytic properties of a plasma kallikrein

Fitzgerald trait, an asymptomatic disorder, is associated with abnormalities of surface-mediated plasma reactions, including coagulation via the intrinsic pathway, augmentation of the clot- promoting properties of factor VII, kaolin-mediated fibrinolysis, kinin generation, and enhancement of vascular permeability by diluted plasma (PF/Dil). These abnormalities can be corrected by Fitzgerald factor, an agent probably identical with high molecular weight kininogen found in normal, but not Fitzgerald-trait plasma. Our preparations of Fitzgerald factor possessed a second property. Amidolysis of alpha-N-benzoyl-L- proline-L-phenylalanine-L-arginine-pnitroanilide by a plasma kallikrein (activated Fletcher factor) was inhibited by kaolin or solutions of ellagic acid. Addition of preparations of Fitzgerald factor to kaolin or to solutions of ellagic acid counteracted their inhibitory properties. The action of these preparations was duplicated by solutions of cytochrome C or IgG, suggesting that these agents may inhibit the negative charges of kaolin or ellagic acid. Fitzgerald factor enhanced amidolysis of both normal and Fitzgerald-trait plasmas exposed to kaolin, effects not duplicated by cytochrome C or IgG. Whether or not the two properties of our preparations of Fitzgerald factor are related to the same agent is not yet certain. The relationship between these observations and the biologic role of Fitzgerald factor remains to be investigated.