Mechanisms for the involvement of high molecular weight kininogen in surface-dependent reactions of Hageman factor.

The mechanisms by which human high molecular weight kininogen (HMKrK) contributes to the surface-dependent activation of the Hageman factor systems have been studied. The ability of various mixtures of purified human Hageman factor (coagulation factor XII), HMrK, prekallikrein, and kaolin to activate coagulation factor XI was determined with factor XIa (activated factor XI) clotting assays. Hageman factor, HMrK and prekallikrein were required for maximal rates of activation of factor XI. A certain optimal mixture of purified Hageman factor, HMrK, prekallikrein, and kaolin gave the same rapid initial rate of activation of purified factor XI as an equivalent aliquot of factor XI-deficient plasma. This suggests that potent, surface-mediated activation of factor XI in plasma is explicable in terms of Hageman factor, HMrK, and prekallikrein. By studying separately some of the surface-dependent reactions involving Hageman factor, it was found that HMrK accelerated by at least an order of magnitude the following reactions: (i) the activation of factor XI by activated Hageman factor; (ii) the activation of prekallikrein by activated Hageman factor; and (iii) the activation of Hageman factor by kallikrein. Stoichiometric rather than catalytic amounts of HMrK gave optimal activation of factor XI. These results are consistent with the hypothesis that HMrK and Hageman factor form a complex on kaolin which renders Hageman factor more susceptible to proteolytic activation by kallikrein and which facilitates the action of activated Hageman factor on its substrate proteins, factor XI and prekallikrein.     

Urinary excretion of Hageman factor (factor XII) and the presence of nonfunctional Hageman factor in the nephrotic syndrome

Acquired deficiencies of functional Hageman factor (factor XII) and prekallikrein, proteins involved in the plasma kinin-generating system, have been previously reported in the nephrotic syndrome. The basis for these changes, however, is not fully understood. We have examined the levels of Hageman factor and prekallikrein by functional and radioimmunoassays in plasmas and urines of 11 patients with the nephrotic syndrome. All 11 patients had decreased titers of plasma Hageman factor activity (mean ± standard deviation (SD), 0.29 ± 0.15 U/ml), but essentially normal titers of immunoreactive Hageman factor (0.88 ± 0.23 U/ml). The ratio of immunoreactive Hageman factor to functional Hageman factor (2.63 ± 0.86) was significantly higher than that in nine control patients (1.08 ± 0.17). Since no circulating anticoagulants against Hageman factor were detected, these data suggest the presence of nonfunctional (altered) Hageman factor in plasmas of patients with the nephrotic syndrome. Urinary excretion of Hageman factor was present in six patients but did not appear to account for the reduced plasma Hageman factor activity. Urinary Hageman factor in one patient had the same size as plasma Hageman factor as assessed by gel filtration and sucrose density gradient centrifugation. The titers of plasma prekallikrein were within the normal range. These studies indicate urinary excretion of Hageman factor and alterations in the functional sites of plasma Hageman factor molecules in the nephrotic syndrome. Whether these changes are related to the pathogenesis of the nephrotic syndrome remains to be determined.     

The Relation of 'Fletcher Factor' to Factors XI and XII

S ummary . Further evidence is presented for the existence of a new coagulation factor which is closely related to Hageman factor (XII) and plasma thromboplastin antecedent, PTA (XI). This factor has been tentatively designated 'Fletcher factor'. Coagulant activity of Fletcher factor was separated from the clotting activity of factors XI and XII by C-M Sephadex column chromatography of intact normal plasma. Other studies showed that the prolonged partial thromboplastin time or plasma recalcification time of Fletcher-deficient plasma could be 'corrected' by prolonged contact with celite, glass, kaolin, or ellagic acid; all are known activators of factor XII. Cytochrome c, known to inhibit the contact activation of factor XII, completely abolished this contact 'correction' of Fletcher-deficient plasma. Thus, the clotting times of plasmas deficient in Fletcher factor (presently found in seven individuals from four unrelated families) are readily corrected by activated factors XII and XI. None of these individuals has any bleeding tendencies.
Fletcher factor activity is deficient in the plasma of newborn infants; the factor is probably produced in the liver and not dependent on vitamin K for its synthesis.     

Preparation, Characterization, and Activation of a Highly Purified Factor XI: Evidence that a Hitherto Unrecognized Plasma Activity Participates in the Interaction of Factors XI and XII

S ummary . Highly purified native factor XI has been prepared from normal plasma using a five step purification scheme. Purified factor XI is essentially free of factors II, V, VII, VIII, IX, X, XII, and Fletcher factor. No plasminogen-plasmin could be detected. Purified factor XI decays rapidly but can be stabilized with human serum albumin. Factor XI migrates between the β and γ globulins on starch block electrophoresis. It has an apparent molecular weight on gel filtration of 210 000. Purified factor XI is activated by weak trypsin. No detectable BAEe esterase activity accompanies this activation. Neither factor XII adsorbed to kaolin nor activated factor XII in solution could activate purified factor XI; both reagents activate factor XI in dilute factor XII deficient plasma. Hence, plasma appears to supply a third activity which facilitates the interaction of factors XI and XII. This activity is shown to be distinct from Fletcher factor.     

Activation of intrinsic coagulation pathway in pre-eclampsia

Disseminated intravascular coagulation, thrombocytopenia, consumption of factors VIII and II, and antithrombin deficiency have been previously demonstrated in pre-eclampsia. However, the precise mechanism responsible for initiation of disseminated intravascular coagulation has not been elucidated. The present study documents activation of the intrinsic coagulation pathway in a patient with severe pre-eclampsia. The studies revealed marked reductions of plasma coagulant activities of all intrinsic pathway factors, i.e., XII, XI, IX, and VIII. In addition, the ratio of plasma factor XII activity to antigen concentration was markedly abnormal, and plasma high-molecular-weight kininogen concentration was diminished. It is suggested that activation of the intrinsic coagulation pathway may be operative in the genesis of disseminated intravascular coagulation in pre-eclampsia.     

Properties of sulfatides in factor-XII-dependent contact activation

Incubation of normal human plasma with low amounts of sulfatides resulted in the initiation of intrinsic coagulation and the appearance of kallikrein activity. The optimal initiation of procoagulant and kallikrein amidolytic activity was dependent on the presence of factor XII, high molecular weight kininogen, and prekallikrein. Since the activated partial thromboplastin clotting times in prekallikrein- deficient plasma approach normal values upon prolonged incubation with kaolin, this phenomenon of autocorrection was studied and found to be even more pronounced in the presence of sulfatides. Autocorrection was essentially completed in 5 min in the presence of sulfatides, whereas a preincubation of 15-20 min was required in the presence of kaolin. The limited proteolysis of 125I-factor XII in plasma during incubation with activating material or during clotting was determined. Cleavage of factor XII was more rapid and more extensive in the presence of sulfatides than in the presence of kaolin. In prekallikrein-deficient plasma, factor XII cleavage was completed within 5 min in the presence of sulfatides and within 15 min in the presence of kaolin. Thus, the appearance of factor-XII-dependent coagulant activity correlates with the limited proteolysis of factor XII when normal or prekallikrein- deficient plasma is activated by sulfatides or kaolin.     

Inhibition of platelet prothrombinase activity by a lupus anticoagulant

Lupus anticoagulants are spontaneously occurring antibodies with specificity for negatively charged phospholipids. The plasma of a patient with such a polyclonal antibody of IgM type demonstrated low levels of factor VIII coagulant activity (VIII:C) and factors IX, XI and XII when analyzed by biologic clotting assays, whereas in immunochemical assays, normal levels of VIII coagulant antigen and factor IX were obtained. After immunoadsorption of patient plasma with anti-IgM Sepharose, normal biologic activities were demonstrated in clotting assays for VIII:C, factors IX, XI, and XII. The addition of the patient's isolated IgM to normal plasma resulted in grossly abnormal results in these coagulation assays, and a pattern similar to that of the patient's plasma was obtained. The inhibitory effect of the patient's lupus anticoagulant on blood coagulation was demonstrated also in platelet-rich plasma. The results of the clotting assays indicated that the anticoagulant inhibited several of the reactions in the blood coagulation cascade. The availability of purified components made it possible to demonstrate an inhibiting effect on the activation of prothrombin by factor Xa in the presence of isolated platelets, as well as in a system where purified factor V and well defined phospholipid vesicles were substituted for the platelets.     

Factor XII auto-antibodies present in a patient with a B-cell lymphoma.

A 64-year-old woman was transferred for investigation of a mediastinal mass, biopsy of which showed a diffuse large B-cell lymphoma. She was also found to have an antiphospholipid antibody. The pre-operative coagulation screen showed a prolonged activated partial thromboplastin time, 71.3 s (normal range, 26-36 s), which was not corrected by the addition of normal plasma. The dilute Russell's viper venom time was positive. Anti-cardiolipin assay was strongly positive, immunoglobulin M was 153 AU; immunoglobulin G was normal, 3.1 AU. Assays of factors VIII, IX and XI showed higher concentrations with increasing dilutions in one-stage factor assays from 1: 10 to 1: 80 suggestive of an inhibitor. Factor XII was 9 U/dl and results were unaffected by increasing dilution, suggesting specific antibodies to factor XII. The factor XII antigen was 40 U/dl. The patient had immunoglobulin M auto-antibodies to factor XII.     

Role of surface in surface-dependent activation of Hageman factor (blood coagulation Factor XII)

The mechanism by which negatively charged substances such as celite, kaolin, or ellagic acid contribute to the surface-dependent activation of Hageman factor (Factor XII) was studied. Kinetic studies of the proteolytic activation of (125)I-labeled human Hageman factor by human plasma kallikrein, plasma, activated Factor XI, and trypsin were performed in the presence and absence of high molecular weight kininogen and surface materials such as celite, kaolin, or ellagic acid. The results showed that surface-bound Hageman factor was 500 times more susceptible than soluble Hageman factor to proteolytic activation by kallikrein in the presence of high molecular weight kininogen. Surface binding of Hageman factor enhanced its cleavage by plasmin, activated Factor XI, and trypsin by 100-fold, 30-fold, and 5-fold, respectively. On a molar basis, trypsin was twice as potent as kallikrein in the cleavage of the surface-bound Hageman factor, while plasmin and activated Factor XI were an order of magnitude less potent than kallikrein. Kallikrein even at concentrations as low as 0.5 nM (i.e., 1/1000th of the concentration of prekallikrein in plasma) was very potent in the limited proteolysis of the surface-bound Hageman factor. These results suggest that substances classically known as "activating surfaces" promote the activation of Hageman factor indirectly by altering its structure such that it is much more susceptible to proteolytic activation by other plasma or cellular proteases.     

The Effects of Collagen and Kaolin on the Intrinsic Coagulant Activity of Platelets. EVIDENCE FOR AN ALTERNATIVE PATHWAY IN INTRINSIC COAGULATION NOT REQUIRING FACTOR XII

Collagen and kaolin have been shown by other workers to initiate intrinsic coagulation by activating factor XII in plasma and to have complex effects on platelets. Because of the presence of collagen at sites of vascular injury there is good reason to believe that collagen has physiological importance in haemostasis. The present experiments were done to determine the effects of collagen and kaolin on platelets and to distinguish the platelet effects from the activity which these surface-active agents produce in plasma.
Using an albumin-density-gradient separation (ADGS) method for washing platelets free of loosely adsorbed coagulation factors, it is shown here that collagen can induce a coagulant activity in platelets which initiates intrinsic coagulation. This activity is independent of factor XII, provided factor XI is present. It is postulated that this collagen-induced coagulant activity of platelets provides an alternative pathway, by-passing factor-XII activation, for initiating intrinsic coagulation. The existence of this alternative pathway may provide an explanation for the absence of a haemostatic defect in Hageman trait. The effects of kaolin were similar to those of collagen, but kaolin had greater capacity to activate plasma factor XII and platelet factor 3 and relatively less capacity to activate platelet-associated factor XI.     

Platelets and Initiation of Intrinsic Clotting

S ummary . Comparison of activities in platelet rich and platelet poor plasmas from normal donors and patients deficient in either factor VIII, IX, XI or XII indicates that platelets contain activities which can partially substitute for plasma factors XI and XII. The factor-XI-like activity is expressed in a one-stage activated partial thromboplastin assay and in an intact prothrombin consumption system. The factor-XII-like activity is scarcely detectable in a one-stage assay but markedly enhances the defective prothrombin consumption of factor XII deficient plasma. Intact prothrombin consumption tests with platelet poor plasmas fortified with cephalin show that in the presence of high concentrations of platelet factor 3 activity only trace contact activation is required to promote good prothrombin consumption. The platelet, by supplying both platelet factor 3 and activities bypassing plasma contact activation factors XI and XII, may provide an important route for activating intrinsic clotting.     

Screening coagulation tests and clotting factors in homozygous beta-thalassemia.

In 30 children with homozygous beta-thalassemia the hemostasis screening tests (bleeding time, PT, PTT), platelet count and specific assays of clotting factors were carried out 25 days after their last transfusion. PT, PTT, and bleeding time showed minor variations; considerable thrombocytosis was found in splenectomized patients. Factors IX and XII were decreased in a high proportion of patients, the vitamin K-dependent factors (II, VII, IX, X) were slightly reduced and factors I, V and VIII remained within the normal range in a majority of patients. Hepatic failure resulting in defective protein synthesis does not explain the more marked impairment of factors XI and XII, which might be secondary to activation of the intrinsic coagulation and/or kallikrein systems following intravascular haemolysis and multiple blood transfusions.     

Activation of factor XI in plasma is dependent on factor XII [see comments]

The activation of factor XI initiates the intrinsic coagulation pathway. Until recently it was believed that the main activator of factor XI is factor XIIa in conjunction with the cofactor high molecular weight kininogen on a negatively charged surface. Two recent reports have presented evidence that in a purified system factor XI is activatable by thrombin together with the soluble polyanion dextran sulfate. To assess the physiological relevance of these findings we studied the activation of factor XI in normal and factor XII-deficient plasma. We used either kaolin/cephalin or dextran sulfate as a surface for the intrinsic coagulation pathway, tissue factor to generate thrombin via the extrinsic pathway, or the addition of alpha-thrombin directly. 125I-factor XI, added to factor XI-deficient plasma at physiologic concentrations (35 nmol/L), is rapidly cleaved on incubation with kaolin. The kinetics appear to be exponential with half the maximum cleavage at 5 minutes. Similar kinetics of factor XI cleavage are seen when 40 nmol/L factor XIIa (equal to 10% of factor XII activation) is added to factor XII-deficient plasma if an activating surface is provided. Tissue factor (1:500) added to plasma did not induce cleavage of factor XI during a 90-minute incubation, although fibrin formation within 30 seconds indicated that thrombin was generated via the extrinsic pathway. Adding 1 mumol/L alpha-thrombin (equivalent to 50% prothrombin activation) directly to factor XII deficient or normal plasma (with or without kaolin/cephalin/Ca2+ or dextran sulfate) led to instantaneous fibrinogen cleavage, but again no cleavage of factor XI was observable. We conclude that in plasma surroundings factor XI is not activated by thrombin, and that proposals of thrombin initiation of the intrinsic coagulation cascade are not supportable.     

Decreased blood coagulation activities in carbohydrate-deficient glycoprotein syndrome

Summary The carbohydrate-deficient glycoprotein (CDG) syndromes are a newly recognized group of inherited metabolic diseases. We report a Japanese brother and sister with a CDG syndrome. Both patients showed decreased activities of blood coagulation Factor XI and of the coagulation inhibitor protein C. In one of them there was also a somewhat decreased activity of Factor IX and of antithrombin III. Isoelectric focusing of antithrombin III revealed a decrease of negatively charged fractions and an increase of more cathodal bands. Furthermore, there was a discrepancy between activity and antigen level of Factor VIII and protein C. The patients had an incidental deficiency of factor XII. This is the first detailed report on blood coagulation systems in the CDG syndromes. These blood coagulation abnormalities may explain at least in part the thrombotic or haemorrhagic complications of the CDG syndromes.     

Factors XI and XII are low in subjects with liver disease

We prospectively measured levels of factors XI and XII in parallel with other coagulation factors in 39 unselected patients with liver disease and in 20 control subjects. Mean levels of factors XI and XII in subjects with liver disease were significantly reduced, being 58% and 61%, respectively, compared with 100% and 94% in controls. Reductions in levels of factors XI and XII were most pronounced in those subjects with low serum albumin. The partial thromboplastin time (APTT) reflected low levels of either factor XI or XII and was most prolonged when both were low, but cause and effect was not demonstrated. Low levels of these factors may explain previous reports of poor response of APTT to infusions of prothrombin complex concentrates. Finally, these low levels strongly suggest that factors XI and XII are produced in the liver.     

Lupus anticoagulant-hypoprothrombinemia syndrome with a false-positive test for coagulation factor inhibitors

We report a case of a 1-year-old boy diagnosed with lupus anticoagulant-hypoprothrombinemia syndrome (LA-HPS), which is a rare disorder. His initial presentation of sinusitis was accompanied by hemorrhagic episodes including ecchymoses and epistaxis 6 months after antibiotic therapy. Laboratory results revealed prolonged prothrombin time (PT) and activated partial thromboplastin time (APTT) that did not correct with mixing studies. Factors II, VIII, IX, X, XI, and XII activities were 20%, 44%, 42.5%, 59%, 4%, and 10%, respectively. The Bethesda inhibitor assay showed inhibitors against multiple coagulation factor. APTT, mixing studies, diluted Russell's viper venom time, and the Bethesda inhibitor assay detected LA. LA-HPS with a suspected false-positive test for coagulation factor inhibitors was diagnosed. Bleeding stopped and results of coagulation studies returned to normal without therapy 2 months after onset of the disease.     

Suppressed intrinsic fibrinolytic activity by monoclonal anti-beta-2 glycoprotein I autoantibodies: possible mechanism for thrombosis in patients with antiphospholipid syndrome

beta2-glycoprotein I (beta2GPI) bears the epitope(s) for autoimmune anticardiolipin antibodies (aCL) frequently present in patients with antiphospholipid syndrome (APS). beta2GPI is involved in coagulation and fibrinolytic systems, including inhibition of contact activation. Coagulation factor XII is an initiator of intrinsic coagulation and also of intrinsic fibrinolysis. We investigated the effect of aCL (= anti-beta2GPI antibodies), regarding intrinsic fibrinolysis using autoimmune monoclonal anti-beta2GPI antibodies derived from a patient with APS or from an NZW/BXSB-F1 mouse. We developed a chromogenic assay system to determine intrinsic fibrinolytic activity. The reaction was activated by kaolin in the euglobulin fraction. Exogenous beta2GPI slightly suppressed intrinsic fibrinolytic activity of the euglobulin fraction from normal plasma. Human monoclonal anti-beta2GPI antibody (EY2C9) and mouse monoclonal anti-beta2GPI antibody (WBCAL-1) in the presence of beta2GPI decreased the activity. In this system, the suppression remained significant in the presence of an excess of exogenous activated factor XII. Euglobulin fractions from APS patients' plasma paralleled low activities of intrinsic fibrinolysis compared with those from healthy subjects. Our results suggest that beta2GPI and anti-beta2GPI antibodies suppress intrinsic fibrinolytic activities. This suppression was not only due to inhibition of factor XII activation but was also related to function of activated factor XII (XIIa). These phenomena partly explain the mechanisms of thrombosis in APS.     

A study of antihaemophilic globulin (factor VIII) consumption.

A study has been made of the rate of disappearance of factor VIII during the clotting of normal plasma samples and of samples deficient in factors V, VII, IX, X, XI and XII. The rate of disappearance (or consumption) of factor VIII was the same in the normal and factor VII deficient samples. Delayed disappearance of factor VIII was observed in samples deficient in factors V, IX, X, XI and XII. The significance of the findings to the theory of blood coagulation is discussed.     

Acquired inhibitors to coagulation factors in patients with gastrointestinal diseases

OBJECTIVES: To study the frequency and specificity of acquired coagulation inhibitors in inflammatory and malignant gastrointestinal diseases. METHODS: In a 10-year period, 511 patients from the island of Crete in Greece were studied, 302 with ulcerative colitis, 112 with Crohn's disease, 82 with gastrointestinal carcinoma and 15 with gastrointestinal lymphoma. Prothrombin time and activated partial thromboplastin time were measured by routine methods. When prothrombin time and/or activated partial thromboplastin time were found to be prolonged, mixture experiments with 25%, 50% and 75% pooled normal human plasma were performed. If clotting times were inadequately corrected, the presence of an acquired inhibitor against a coagulation factor was suggested. Specific coagulation factor assays were then performed with deficient plasmas. RESULTS: Fifteen patients acquired inhibitors to the following coagulation factors within the 10-year observation period: factor IX (four patients); factor X (three patients); factor XII (three patients); factor VIII (two patients); factor XI (two patients); and factor V (one patient). The activity of the above factors varied from < 1% to 10%. Five patients with ulcerative colitis, six with Crohn's disease, two with gastrointestinal lymphoma and two with gastrointestinal carcinoma developed an inhibitor. Only one patient with factor VIII inhibitor presented with severe bleeding and was treated with recombinant human activated factor VII, while the others had no complications. Remission was obtained in all patients after immunosuppressive therapy, chemotherapy or tumour resection. CONCLUSION: An increased incidence of coagulation factor inhibitors was found in patients with gastrointestinal inflammatory and malignant diseases compared to the healthy population. In addition, an increased incidence of these inhibitors was also found in the common population of Crete compared to that found in other areas.     

Plasma contact factors as therapeutic targets

Direct oral anticoagulants (DOACs) are small molecule inhibitors of the coagulation proteases thrombin and factor Xa that demonstrate comparable efficacy to warfarin for several common indications, while causing less serious bleeding. However, because their targets are required for the normal host-response to bleeding (hemostasis), DOACs are associated with therapy-induced bleeding that limits their use in certain patient populations and clinical situations. The plasma contact factors (factor XII, factor XI, and prekallikrein) initiate blood coagulation in the activated partial thromboplastin time assay. While serving limited roles in hemostasis, pre-clinical and epidemiologic data indicate that these proteins contribute to pathologic coagulation. It is anticipated that drugs targeting the contact factors will reduce risk of thrombosis with minimal impact on hemostasis. Here, we discuss the biochemistry of contact activation, the contributions of contact factors in thrombosis, and novel antithrombotic agents targeting contact factors that are undergoing pre-clinical and early clinical testing.