The conversion of C'IS to C'1 esterase by plasmin and trypsin.

The formation of C'1 esterase from C'1, the first component of complement, may be brought about by the action of plasmin or trypsin upon C'1s, a subcomponent of C'1. These enzymes also decrease the esterolytic activity of C'1 esterase. The formation of C'1 esterase was demonstrated by measuring the appearance of an agent or agents with esterolytic properties and the capacity to inactivate C'2 and C'4, attributes of C'1 esterase. The activity of the agent which evolved was blocked by serum inhibitor of C'1 esterase. The implications of these observations, that the formation of C'1 esterase during complement fixation is mediated by proteolytic processes, are under study. The possible inhibition of C'1q by soybean trypsin inhibitor is in agreement with this hypothesis.

The inhibition of plasmin, plasma kallikrein, plasma permeability factor, and the C'1r subcomponent of the first component of complement by serum C'1 esterase inhibitor.

The fraction of human serum designated as C'1 esterase inhibitor is known to inhibit the action of C'1 esterase, a plasma kallikrein, and PF/Dil, an enzyme in plasma enhancing cutaneous vascular permeability. In the present study, C'1 esterase inhibitor has been found to block the actions of plasmin and the C'1r subcomponent of the first component of complement, and to retard the generation of PF/Dil. No inhibition of blood clotting or of the generation of plasmin was demonstrable.

Activation of Hageman factor by L-homocystine.

L-Homocystine activates Hageman factor, as demonstrated by its capacity to initiate clotting and to induce the evolution of plasma kinins. Perhaps, strategically located deposits of this amino acid are responsible for the unusual frequency of thrombosis in patients with homocystinuria.

Immunologic studies in von Willebrand's disease. Evidence that the antihemophilic factor (AHF) produced after transfusions lacks an antigen associated with normal AHF and the inactive material produced by patients with classic hemophilia.

Antihemophilic globulin (AHF, factor VIII) levels were measured by a standard coagulation assay and by an immunological technique before and serially after infusion of fresh frozen plasma or cryoprecipitate into patients with von Willebrand's disease. Initial levels of AHF, measured both as procoagulant and as antigen, were low. Immediately after transfusions, the rise in levels of AHF-like antigen was compatible with the quantity of antigen present in the infused plasma or cryoprecipitate. Thereafter, levels of antigen declined rapidly and reached preinfusion values in approximately 24 hr. In contrast, procoagulant activity remained elevated, and sometimes continued to rise, for longer periods of time. One possible explanation of this finding is that the AHF molecule produced by patients with von Willebrand's disease, in response to transfusion of as yet unidentified factors, lacks the antigenic site associated with the normal AHF molecule or the inactive molecule produced by patients with hemophilia A.

Studies on the response of patients with classic hemophilia to transfusion with concentrates of antihemophilic factor. A difference in the half-life of antihemophilic factor as measured by procoagulant and immunologic techniques.

Antihemophilic factor (AHF, factor VIII) levels were measured by a standard coagulation method and by an immunologic technique before and after infusion of AHF concentrates into patients with classic hemophilia. After infusion of AHF concentrates, the half-life of the AHF procoagulant (i.e., clot-promoting) activity varied from 12 to 14 hr, whereas that of the antigen ranged from 24 to 40 hr. The half-life of the antigen was similar in patients with and without circulating anticoagulants to AHF. The data are compatible with the suggestion that the antigen may be carried on a precursor molecule which the patient with hemophilia produces but cannot convert to the functional clot-promoting agent. Other explanations of the observations are, however, recognized.

Inhibition of the activation of Hageman factor (factor XII) by peripheral blood cells.

Suspensions of peripheral blood mononuclear cells (PBMC), monocytes, T or B lymphocytes, platelets or granulocytes, and cell-depleted supernatant fluids of these suspensions inhibited activation of Hageman factor (HF, Factor XII) by ellagic acid, a property not shared by erythrocytes. PBMC also inhibited HF activation by glass or sulfatides. Contaminating platelets may have contributed to inhibition by PBMC. Elaboration of agents inhibiting HF activation required metabolically active cells. The inhibitor(s) in PBMC supernates were not identified with known agents, but had properties of a nonenzymatic protein. PBMC supernates did not contain fibrinogen, nor alter the thrombin, prothrombin, or partial thromboplastin times of normal plasma, amidolysis by activated plasma thromboplastin antecedent (Factor XIa) or activated Stuart factor (Factor Xa) or esterolysis by C1 (C1 esterase); they inhibited plasmin minimally. These experiments suggest that peripheral blood cells may impede intravascular coagulation. Whether this property helps maintain the fluidity of blood is unclear.

Studies on the purification of antihemophilic factor (factor 8. I. Precipitation of antihemophilic factor by concanavalin A.

Concanavalin A precipitates antihemophilic factor from normal plasma. Combining this precipitation with other techniques, we were able to separate fractions rich in antihemophilic activity from human plasma with rapidity. The molecular weight of antihemophilic factor was estimated to be greater than 2 million. Presumably, antihemophilic factor is a large glycoprotein.

Studies on the purification of antihemophilic factor (factor VIII). II. Separation of partially purified antihemophilic factor by gel filtration of plasma.

A high degree of purification of antihemophilic factor was achieved by filtration of chylomicronpoor human plasma through columns of agarose. The final product contained, on the average, 67 units of antihemophilic activity per mg of protein, and was 3360-fold purified compared with the filtered plasma. The molecular weight of antihemophilic factor appeared to be at least two million. Preparations separated by gel filtration were contaminated with appreciable amounts of plasma thromboplastin antecedent (PTA), and traces of Christmas factor and Hageman factor, but no detectable fibrinogen was present. Similar fractions of plasma prepared from the blood of patients with classic hemophilia, von Willebrand's disease, or a circulating anticoagulant directed against antihemophilic factor contained, on the average, somewhat less protein than normal plasma; whether this difference was significant is not yet known. The purified fractions were partially stabilized by the addition of 1% gelatin. Adaptation of the technique of gel filtration to purification of antihemophilic factor for clinical use remains to be explored.

Studies on the nature of antihemophilic factor (factor VIII). Further evidence relating the AHF-like antigens in normal and hemophilic plasmas.

Normal human antihemophilic factor (AHF, factor VIII) and the protein antigenically related to it in hemophilic plasma both appeared in the void volume of columns of agarose (Sepharose 4B) during purification of these agents. On ultracentrifugation upon sucrose gradients, both agents had sedimentation characteristics similar to those of an S30 marker. After reduction, the polypeptide chains of purified normal AHF and of the nonfunctional agent from hemophilic patients had an apparent molecular weight of 200,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These observations suggest that AHF, purified as described, exists as a large molecule with subunits of molecular weight of approximately 200,000. Antisera to normal AHF and the nonfunctional agent from hemophilic plasma appeared to be directed against antigens of similar electrophoretic mobility and precipitating characteristics, present in normal and hemophilic plasma but deficient in severe von Willebrand's disease plasma. Both antisera neutralized the AHF clot-promoting activity present in normal plasma, and this property was removed by absorption of the antisera with concentrates of normal or hemophilic plasma but to a greatly reduced extent by concentrates of von Willebrand's disease plasma. These findings suggest that the antigen detected in normal plasma by the antisera appears on a molecule participating in the AHF clot-promoting reaction.

Influence of augmented Hageman factor (Factor XII) titers on the cryoactivation of plasma prorenin in women using oral contraceptive agents.

Prolonged cold storage of plasma may induce the conversion of plasma prorenin (inactive renin) to renin. This phenomenon is exaggerated in oral contraceptive (OC) users; the titer of Hageman factor (HF, Factor XII) in OC users is higher than in nonusers. The present study relates these observations. The increment in plasma renin activity (PRA) during cold storage, as measured by generation of angiotensin I, correlated strongly with the initial plasma titer of HF. Increasing the HF titer of nonusers to that observed in OC users by addition of purified HF increased cold-induced PRA at least twofold, while reducing the plasma HF titer of OC users correspondingly decreased cold-induced PRA. Thus, in OC users, the enhanced conversion of plasma prorenin to renin during cold storage reflects the elevated plasma titer of HF.

PIP: Prolonged cold storage of plasma may induce the conversion of plasma prorenin (inactive renin) to renin. This phenomenon is exaggerated in oral contraceptive (OC) users; the titer of Hageman factor (HF, Factor 12) in OC users is higher than in nonusers. The present study relates these observations. The increment in plasma renin activity (PRA) during cold storage, as measured by generation of angiotensin I, correlated strongly with the initial plasma titer of HF. Increasing the HF titer of nonusers to that observed in OC users by the addition of purified HF increased cold-induced PRA at least 2-fold, while reducing the plasma HF titer of OC users correspondingly decreased cold-induced PRA. Thus, in OC users, the enhanced conversion of plasma prorenin to renin during cold storage reflects the elevated plasma titer of HF.

Immunologic differentiation of classic hemophilia (factor 8 deficiency) and von Willebrand's dissase, with observations on combined deficiencies of antihemophilic factor and proaccelerin (factor V) and on an acquired circulating anticoagulant against antihemophilic factor.

Heterologous antiserum was prepared in rabbits against highly purified human antihemophilic factor (AHF, factor VIII). This antiserum blocked the clot-promoting properties of AHF and, when suitably absorbed, formed a single precipitin line against AHF upon immunoelectrophoresis. Material antigenically similar to normal AHF was detected in normal amounts in plasma concentrates in each of 22 patients with classic hemophilia, in a patient with an acquired circulating anticoagulant against AHF, and in a patient with deficiencies both of AHF and proaccelerin (factor V). AHF-like antigen was present in normal human serum. In contrast, material antigenically related to AHF was found in decreased amounts in the concentrates prepared from the plasma of 11 patients with von Willebrand's disease. The experiments described suggest that von Willebrand's disease is a disorder in which a true deficiency of AHF exists. Whether the AHF-like material found in classic hemophilia is nonfunctional through a defect in structure or through the intervention of an inhibitor has not been shown.

Detection of carriers of classic hemophilia using an immunologic assay for antihemophilic factor (factor 8().

The relation between functional antihemophilic factor (AHF) activity and AHF-like antigen was studied in the plasma of 25 known carriers of hemophilia. In 23 cases, this relationship was significantly different from that in normal women, at the 99% limit of confidence. In contrast, among families in which only one case of hemophilia had occurred, only five of nine mothers could be identified as carriers. This observation suggests that in some instances the hemophilia arose from a newly mutant gene. The data are consistent with the hypothesis that the proportion of antigen to AHF activity in carriers is determined by random activation or inactivation of the X chromosome.

Fitzgerald Trait: Deficiency of a Hitherto Unrecognized Agent, Fitzgerald Factor, Participating in Surface-Mediated Reactions of Clotting, Fibrinolysis, Generation of Kinins, and the Property of Diluted Plasma Enhancing Vascular Permeability (PF/Dil).

The prolonged partial thromboplastin time observed in the plasma of a 71-yr-old asymptomatic man was related to the deficiency of a hitherto unrecognized agent. The patient's plasma also exhibited impaired surface-mediated fibrinolysis and esterolytic activity and impaired generation of kinins and of the property enhancing vascular permeability designated PF/Dil. The patient's plasma contained normal amounts of all known clotting factors except Fletcher factor (a plasma prekallikrein) which was present at a concentration of 10-15% of pooled normal plasma. Fletcher trait plasma, however, contained normal amounts of the agent missing from the patient's plasma and corrected the defects in clotting, fibrinolysis, and vascular permeability. Fletcher trait plasma was less effective in correcting generation of kinins and esterolytic activity, presumably because of the patient's partial deficiency of prekallikrein. The site of action of the factor deficient in the patient's plasma appeared to be subsequent to the activation of Hageman factor and plasma prekallikrein. A fraction of normal plasma, devoid of other clotting factors, corrected the defect in clotting in the patient's plasma; a similar fraction of the patient's plasma did not correct this abnormality. No evidence yet exists pointing to the familial nature of the patient's defect. Tentatively, the patient's disorder may be referred to by his surname as Fitzgerald trait, and the agent apparently deficient in his plasma as Fitzgerald factor.

Inhibition of the activation of Hageman factor (factor XII) by complement subcomponent C1q.

Hageman factor (HF, Factor XII) is activated by glass, collagen, and ellagic acid, and initiates blood coagulation via the intrinsic pathway. C1q inhibits collagen-induced platelet aggregation and adherence of platelets to glass, effects attributable to the collagen-like region of C1q. We examined the actions of C1q on HF activation. Incubation of C1q with HF before addition of HF-deficient plasma extended the activated partial thromboplastin time. Similarly, when glass tubes were coated with C1q before testing, the partial thromboplastin time of normal plasma was increased. C1q reduced the activation of HF by ellagic acid, as measured by the release of p-nitroaniline from the synthetic substrate H-D-prolyl-L-phenylalanyl-L-arginine-p-nitroanilide dihydrochloride, an effect inhibited by monoclonal anti-human C1q murine IgG and by digestion of C1q by collagenase. Thus, C1q inhibits activation of HF in vitro in clot-promoting and amidolytic assays and suggests a regulatory mechanism for the inhibition of coagulation.

Fibrinogen Cleveland II. An abnormal fibrinogen with defective release of fibrinopeptide A.

An abnormal fibrinogen (fibrinogen Cleveland II) was detected in the plasma of a 23-yr-old white man with a mild bleeding diathesis. The one-stage prothrombin time, thrombin time, and Reptilase time were all prolonged. 16 of 24 tested relatives had the defect, which appeared to be transmitted as an autosomal dominant characteristic. The thrombin time of normal plasma was slightly inhibited by the proband's plasma. The abnormally long thrombin time of fibrinogen Cleveland II was partially corrected by addition of calcium ions. Fibrinogen Cleveland II was indistinguishable from normal fibrinogen by immunoelectrophoresis, DEAE-cellulose column chromatography, or polyacrylamide gel electrophoresis of reduced fibrinogen in sodium dodecyl sulfate. The major defect detected appeared to be impaired release of fibrinopeptide A when fibrinogen Cleveland II was incubated with thrombin. This defect was localized to the NH(2)-terminal disulfide knot portion of the molecule. An abnormality of polymerization of fibrin monomers was also present, but the abnormal fibrin demonstrated relatively normal crosslinking. Despite these defects, fibrinogen Cleveland II achieved a degree of coagulability similar to normal fibrinogen and appeared to incorporate some molecules of fibrin with intact fibrinopeptide A into the clot. The fibrin clot that was formed appeared to be abnormal by electron microscopy. These functional defects and other descriptive characteristics appear to distinguish fibrinogen Cleveland II from other inherited abnormal fibrinogens.

The secondary structure of human Hageman factor (factor XII) and its alteration by activating agents.

Hageman factor (factor XII) is activated by exposure to surfaces such as glass or by solutions of certain compounds, notably ellagic acid. Changes in the structure of Hageman factor accompanying activation have been examined in this study by circular dichroism spectroscopy. The spectrum of unactivated Hageman factor in aqueous solutions suggests that its conformation is mainly aperiodic. Various perturbants altered the conformation of Hageman factor in differing ways, demonstrating the sensitivity of Hageman factor to its environment. After activation of Hageman factor with solutions of ellagic acid, a negative trough appeared in the region of the circular dichroism spectrum commonly assigned to tyrosine residues, along with other minor changes in the peptide spectral region. Some of these changes are similar to changes that occurred upon partial neutralization of the basic residues at alkali pH. Activation of Hageman factor by adsorption to quartz surfaces (in an aqueous environment) also produced changes similar to those in the ellagic acid-activated Hageman factor, including the negative ellipticity in the tyrosine region. These observations suggest that the activation process may be related to a change in status of some of the basic amino acid residues, coupled with a specific change in the environment of some tyrosine residues. The importance of these changes during the activation process remains to be determined. The sensitivity of Hageman factor to its environment is consistent with the view that the initiation of clotting by exposure of plasma to appropriate agents is brought about by alterations in the conformation of Hageman factor that occur in the apparent absence of Fletcher factor or other recognized clotting factors.

Studies on a complex mechanism for the activation of plasminogen by kaolin and by chloroform: the participation of Hageman factor and additional cofactors.

As demonstrated by others, fibrinolytic activity was generated in diluted, acidified normal plasma exposed to kaolin, a process requiring Hageman factor (Factor XII). Generation was impaired by adsorbing plasma with glass or similar agents under conditions which did not deplete its content of Hageman factor or plasminogen. The defect could be repaired by addition of a noneuglobulin fraction of plasma or an agent or agents eluted from diatomaceous earth which had been exposed to normal plasma. The restorative agent, tentatively called Hageman factor-cofactor, was partially purified by chromatography and had an apparent molecular weight of approximately 165,000. It could be distinguished from plasma thromboplastin antecedent (Factor XI) and plasma kallikrein, other substrates of Hageman factor, and from the streptokinase-activated pro-activator of plasminogen. Evidence is presented that an additional component may be needed for the generation of fibrinolytic activity in mixtures containing Hageman factor, HF-cofactor, and plasminogen.The long-recognized generation of plasmin activity in chloroform-treated euglobulin fractions of plasma was found to be dependent upon the presence of Hageman factor. Whether chloroform activation of plasminogen requires Hageman factor-cofactor was not determined, but glass-adsorbed plasma, containing Hageman factor and plasminogen, did not generate appreciable fibrinolytic or caseinolytic activity. These studies emphasize the complex nature of the mechanisms which lead to the generation of plasmin in human plasma.

Permeability-increasing activity in hereditary angioneurotic edema plasma. II. Mechanism of formation and partial characterization.

Plasma from persons with hereditary angioneurotic edema readily developed the capacity to increase vascular permeability and to induce the isolated rat uterus to contract. Both activities resided in a small, heat-stable molecule that was apparently a polypeptide. Crude preparations of the polypeptide were inactivated during incubation with trypsin. They also failed to produce pain and erythema, but caused markedly increased vascular permeability in human skin. These characteristics differ from those of bradykinin, from which crude preparations of the polypeptide could also be distinguished by electrophoretic mobility and paper chromatographic behavior. Proof that the polypeptide is truly different from bradykinin must await its further purification. Histamine played no role in the activities observed. Although the enzymes functioning to release the permeability factor and kinin activities in hereditary angioneurotic edema plasma were not clearly defined, one or more plasma enzymes other than C'1 esterase presumably participated either in conjunction with C'1 esterase or in pari passu events to release the polypeptide mediating these activities.

Partial purification of plasma thromboplastin antecedent (factor XI) and its activation by trypsin.

A persistent puzzle in our understanding of hemostasis has been the absence of hemorrhagic symptoms in the majority of patients with Hageman trait, the hereditary deficiency of Hageman factor (factor XII). One proposed hypothesis is that alternative mechanisms exist in blood through which plasma thromboplastin antecedent (PTA, factor XI) can become active in the absence of Hageman factor. In order to test this hypothesis, the effect of several proteolytic enzymes, among them thrombin, plasma kallikrein, and trypsin, was tested upon unactivated PTA. PTA was prepared from normal human plasma by Ca(3)(PO(4))(2) adsorption, ammonium sulfate fractionation, and successive chromatography on QAE-Sephadex (twice). Sephadex-G150, and SP-Sephadex. The partially purified PTA was almost all in its native form, with a specific activity of 45-70 U/mg protein; the yield was about 10%. It contained no measurable amounts of other known clotting factors, plasmin, plasminogen, nor IgG. Incubation of PTA with trypsin generated potent clot-promoting activity that corrected the abnormally long clotting time of plasma deficient in Hageman factor or PTA but not in Christmas factor. This clot-promoting agent behaved like activated PTA on gel filtration (apparent molecular weight: 185,000) and was specifically inhibited by an antiserum directed against activated PTA. These data suggested that PTA can be converted into its active form by trypsin. PTA was not activated by thrombin, chymotrypsin, papain, ficin, plasmin, plasma kallikrein, tissue thromboplastin, or C. Trypsin converted PTA to its active form enzymatically. Whether trypsin serves to activate PTA in vivo is not yet clear.

Inherited incomplete deficiency of the fourth component of complement (C4) determined by a gene not linked to human histocompatibility leukocyte antigens.

We have studied a family in which the proband had systemic lupus erythematosus and selective incomplete deficiency of the fourth component of complement (C4) (2-5% of the normal level). An additional six healthy family members also had low C4 levels (2.4-24.1% of normal) but no evidence of lupus. This form of inherited C4 deficiency differs from that in previously reported families in that inheritance was autosomal dominant (rather than recessive), C4 levels were markedly reduced (but not undetectable), and there was no linkage to HLA, BF, or C4 structural loci, all known to be within the major histocompatibility complex.