Fibrinolytic studies in 13 unrelated families with factor XII deficiency

BACKGROUND AND METHODS. We report the results of extensive "in vitro" fibrinolytic studies in 18 homozygous and 14 obligatory heterozygous subjects belonging to 13 unrelated families with factor XII deficiency. All homozygotes had unmeasurable factor XII activity (XII:C) and antigen (XII:Ag). None had bleeding symptoms, whereas a myocardial infarction occurred in one of them at age 51. In heterozygotes XII:C and XII:Ag were 55.9 +/- 14.1% and 52.1 +/- 16.4% (corresponding figures in 40 normals 100.6 +/- 18.3% and 101.5 +/- 29.7%). Total intrinsic fibrinolytic activity was assayed on fibrin plates in the dextran sulfate euglobulin fraction of plasma from resting subjects, to which flufenamate was added to inhibit blood plasminogen activator inhibitors. RESULTS. Fibrinolytic activity was reduced in all homozygotes (40 +/- 12 BAU/ml) in comparison to heterozygotes (103 +/- 12 BAU/ml) and normals (98 +/- 20 BAU/ml). The addition of purified activated beta-XII led to a complete restoration of fibrinolysis in homozygotes. The addition of anti-urokinase antibodies completely suppressed the reduced intrinsic fibrinolytic activity in homozygotes (4 +/- 7 BAU/ml), whereas a reduction to about 50% was evident in heterozygotes and normals. CONCLUSIONS. Our data confirm that reduced "in vitro" intrinsic fibrinolytic activity is a common finding in homozygous factor XII deficiency and that two independent mechanisms, one factor XII-dependent and urokinase-independent and the other factor XII-independent and urokinase-dependent, are responsible for the generation of intrinsic fibrinolysis in human plasma.     

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.     

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.     

Identifying hypocoagulable states with a modified global assay of overall haemostasis potential in plasma.

To test the sensitivity of the global assay of overall haemostasis potential (OHP) in detecting hypocoagulation, the OHP was assayed in plasma containing exogenous thrombin (0.04 IU/ml), tissue-plasminogen activator (330 ng/ml), Ca and a platelet reagent. Commercial plasmas with factor II, V, VIII, IX, X, XI, XII or VII deficiency were mixed with normal plasma in different proportions to imitate different severities. Samples from patients with haemophilia and factor XII deficiency were also examined. No clot was found in the absence of factor II/factor X, indicating that the tiny dose of thrombin worked solely as a trigger for the intrinsic pathway activation. Changed levels of the investigated coagulants, apart from factor XII, influenced the outcome. OHPs were decreased in patients with haemophilia but were unchanged or even increased in those with factor XII deficiency. This modified OHP method may therefore be useful for estimating the bleeding tendency in haemophilic patients and to find suitable doses and intervals for prophylactic treatment. It may also be of use in investigations of the effect of antifibrinolytic drugs as well as for identifying a thrombotic tendency in patients with factor XII deficiency. For detection of other coagulation factor deficiencies, our investigations with the commercial plasmas suggest that the OHP assay is also valuable, especially when the intrinsic pathway of the coagulation cascade is impaired.     

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.     

Mitogenic effects of coagulation factor XII and factor XIIa on HepG2 cells.

The structure of coagulation factor XII (Hageman factor), inferred from its DNA sequence, includes two epidermal growth factor (EGF)-homologous domains in its amino-terminal region. This suggests that factor XII may exhibit EGF-like activities. Reciprocal antigenic cross-reactivity between factor XII and EGF was shown by exposing purified human factor XII or mouse EGF to anti-mouse EGF or anti-human factor XII. Western blot analysis showed that anti-mouse EGF recognized intact factor XII at 80 kDa. Together, these results suggest that the EGF-homologous domains are accessible for anti-EGF binding in native factor XII. To determine whether factor XII has mitogenic activity, HepG2 or L cells (10(4) cells per well) were grown in serum-free medium in the presence or absence of factor XII or kaolin-activated factor XII (factor XIIa). Both factors XII and XIIa (6.0 micrograms/ml) enhanced cell proliferation by approximately 2-fold (P less than 0.001 and P less than 0.005, respectively). In contrast, L cells, which are not EGF target cells, were not affected by either factor XII or factor XIIa. Various doses of factor XII enhanced cell proliferation, [3H]thymidine incorporation, and [3H]leucine incorporation in HepG2 cells cultured under the same conditions. These data indicate that factor XII, like EGF, is a mitogen for HepG2 cells and suggest a possible autocrine role in the liver.     

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.     

Histidine-rich glycoprotein binds factor XIIa with high affinity and inhibits contact-initiated coagulation

Histidine-rich glycoprotein (HRG) circulates in plasma at a concentration of 2μM and binds plasminogen, fibrinogen, and thrombospondin. Despite these interactions, the physiologic role of HRG is unknown. Previous studies have shown that mice and humans deficient in HRG have shortened plasma clotting times. To better understand this phenomenon, we examined the effect of HRG on clotting tests. HRG prolongs the activated partial thromboplastin time in a concentration-dependent fashion but has no effect on tissue factor-induced clotting, localizing its effect to the contact pathway. Plasma immunodepleted of HRG exhibits a shortened activated partial thromboplastin time that is restored to baseline with HRG replenishment. To explore how HRG affects the contact pathway, we examined its binding to factors XII, XIIa, XI, and XIa. HRG binds factor XIIa with high affinity, an interaction that is enhanced in the presence of Zn2(+), but does not bind factors XII, XI, or XIa. In addition, HRG inhibits autoactivation of factor XII and factor XIIa-mediated activation of factor XI. These results suggest that, by binding to factor XIIa, HRG modulates the intrinsic pathway of coagulation, particularly in the vicinity of a thrombus where platelet release of HRG and Zn2(+) will promote this interaction.     

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.     

Interactions among Hageman factor, plasma prekallikrein, high molecular weight kininogen, and plasma thromboplastin antecedent.

To investigate the earliest steps of the intrinsic clotting pathway, Hageman factor (Factor XII) was exposed to Sephadex gels to which ellagic acid had been adsorbed; Hageman factor was then separated from the gels and studied in the fluid phase. Sephadex-ellagic acid-exposed Hageman factor, whether purified or in plasma, activated plasma thromboplastin antecedent, but only when high molecular weight kininogen was presnet. In the absence of plasma prekallikrein, maximal activation of plasma thromboplastin antecedent was slightly delayed in plasma, a delay not observed with similarly treated purified Hageman factor. Thus, high molecular weight kininogen was needed for expression of Hageman factor's clot-promoting properties and plasma prekallikrein played a minor role in the interaction of ellagic acid-treated Hageman factor and plasma thromboplastin antecedent.     

Factor XII Tenri, a novel cross-reacting material negative factor XII deficiency, occurs through a proteasome-mediated degradation.

A homozygous cross-reacting material negative factor XII-deficient patient with 3% antigen and activity levels of factor XII was screened for the identification of a mutation at the genomic level. Low-ionic strength single-stranded conformation polymorphism (SSCP) analysis and sequence analysis showed that the proband's gene for factor XII had an A-->G substitution at nucleotide position 7832 in exon 3, resulting in a Tyr34 to Cys substitution in the NH2-terminal type II domain of factor XII. We designated this mutation as factor XII Tenri. Mutagenic polymerase chain reaction (PCR), followed by KpnI digestion, showed a homozygous mutation in the proband's gene and heterozygous mutations in his parents and sister. Immunoprecipitation and Western blot analyses of plasma samples from the factor XII Tenri family indicated that the proband had a trace amount of variant factor XII with an apparent molecular mass of 115 kD, which was converted to the normal 80-kD form after reduction, suggesting that factor XII Tenri was secreted as a disulfide-linked heterodimer with a approximately 35-kD protein, which we identified as alpha1-microglobulin by immunoblotting. Pulse-chase experiments using baby hamster kidney (BHK) cells showed that Tenri-type factor XII was extensively degraded intracellularly, but the addition of cystine resulted in increased secretion of the mutant. Using membrane-permeable inhibitors, we observed that the degradation occurred in the pre-Golgi, nonlysosomal compartment and a proteasome appeared to play a major role in this process. On the basis of these in vitro results, we speculate that the majority of the factor XII Tenri is degraded intracellularly through a quality control mechanism in the endoplasmic reticulum (ER), and a small amount of factor XII Tenri that formed a disulfide-linked heterodimer with alpha1-microglobulin is secreted into the blood stream.     

Symptomatic Combined Homozygous Factor XII Deficiency and Heterozygous Factor V Leiden

A family with a combined deficiency of factor XII and factor V Leiden is presented. The proposita is a 72-year-old who showed a mild to moderate thrombotic tendency characterized by two episodes of deep venous thrombosis and superficial phlebitis between the age of 50 and 71. She was shown to be carrier of homozygous factor XII deficiency and heterozygous FV Leiden mutation. A sister of the proposita showed the same pattern but remained asymptomatic. Other family members showed either isolated heterozygous factor XII deficiency or combined heterozygous factor XII deficiency and heterozygous FV Leiden mutation but were all asymptomatic. These data lend support to those who maintain that FV Leiden is a mild genetic determinant for thrombosis. The role of FXII deficiency as an additional risk factor remains questionable.     

Activation of the coagulation system in polycythemia vera.

Thrombosis is one of the major complications of polycythemia vera. Seventeen patients with polycythemia vera in good hematologic control were evaluated for abnormalities of the coagulation system. Activation of the intrinsic coagulation cascade was suggested by low levels of factor XII, prekallikrein, and kallikrein inhibitors in 12 of 17 patients. The group also demonstrated a significant increase in soluble fibrin complexes using plasma gel filtration on 4% agarose. Fibrin degradation products were normal and antithrombin III levels were slightly elevated. It appears that patients with polycythemia vera have chronic activation of the coagulation system, probably initiated by activation of factor XII. No correlation between the degree of coagulation abnormalities and thromboembolic complications was evident in this group of patients.     

The Role of Platelets in Intrinsic Factor-Xa Formation

Although the essential role of phospholipid in the interaction of factors VII and IX has been demonstrated (Lundblad & Davie, 1964), detailed studies of platelet activity in intrinsic coagulation have not previously been reported. A basic test system has been developed for studying the interaction of factors XIa, VIII, IX, and X in the formation of factor Xa. Using platelets from normal and coagulation-factor-deficient donors, washed by albumin density gradient separation (ADGS), a comparative study of platelets and phospholipid in intrinsic factor-Xa formation has been done under various test conditions.
Both platelets and phospholipid (Folch) are shown here to play a part in intrinsic factor-Xa formation, whereas neither enhances extrinsic factor-Xa formation. Collagen promotes a platelet coagulant activity which increases the rate of factor-Xa formation in the test system employed. The effect of collagen depends entirely on the presence on the platelet surface of factor XI, but not of factor XII. In contrast to collagen, adenosine diphosphate has no effect on this intrinsic platelet coagulant activity. In experiments carried out in the presence of naturally-occurring inhibitors to active clotting factors (anti-factors Ila, Xa, and XIa) it is shown here that contact product is adsorbed to the platelet surface where it is protected from destruction by antifactor XIa. Factor Xa is formed subsequently by the interaction of intrinsic clotting factors, which is presumed to occur on the platelet surface since factor Xa is found in association with the platelets, not the surrounding plasma.     

Inhibition of the activation of Hageman factor (factor XII) by human vascular endothelial cell culture supernates.

The supernatant fluid (conditioned medium) of cultured human vascular endothelial cells inhibits activation of Hageman factor (factor XII), whether by ellagic acid, bovine brain sulfatides, or bismuth subgallate; inhibition appears to be a property of one or more proteins in the culture supernates. This phenomenon may contribute to maintaining the fluidity of circulating blood by inhibiting surface activation of the intrinsic pathway of coagulation.     

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.     

Factor XII deficiency with systemic lupus erythematosus. Biological implications.

A patient with Factor XII (Hageman) deficiency and fulminant systemic lupus erythematosus is presented. The Factor XII deficiency was noted prior to the onset of clinical systemic lupus erythematosus and persisted throughout the patient's course without associated hemorrhagic manifestations. There was no evidence for a circulating anticoagulant. The patient had a rapidly progressive fatal course unresponsive to corticosteroid therapy. Factor XII levels did not increase during therapy with steroids. Despite absence of Hageman factor, evidence for activation of complement by the classic pathway and thromboembolic phenomenon was observed. The role of Factor XII in coagulation and inflammatory pathways and the influence of the factor deficiency on the course of the patient's illness are discussed.     

Intrinsic pathway of coagulation and arterial thrombosis

Formation of a fibrin clot is mediated by a group of tightly regulated plasma proteases and cofactors. While this system is essential for minimizing blood loss from an injured blood vessel (hemostasis), it also contributes to pathologic fibrin formation and platelet activation that may occlude vessels (thrombosis). Many antithrombotic drugs target key elements of the plasma coagulation mechanism such as thrombin and factor Xa, based on the premise that plasma elements contributing to thrombosis are primarily those involved in hemostasis. Recent studies with genetically altered mice raise questions about this paradigm. Deficiencies of the intrinsic pathway proteases factor XII and factor XI are not associated with abnormal hemostasis in mice, but impair formation of occlusive thrombi in arterial injury models, indicating that pathways not essential for hemostasis participate in arterial thrombosis. If factor XII or factor XI make similar contributions to thrombosis in humans, these proteases could be ideal targets for drugs to treat or prevent thromboembolic disease with minimal risk of therapy-associated bleeding.     

Polyphosphates: a link between platelet activation, intrinsic coagulation and inflammation?

Early evidence has already shown that activated platelets play a role in the intrinsic pathway of coagulation, but the mechanism remained unclear. The finding that the dense granules of platelets contain polyphosphate (polyP), which has procoagulant effects, resolved this missing link between activated platelets and coagulation. PolyP secreted from platelets leads via activation of Factor XII to in vivo thrombin and fibrin formation. The relative importance of polyP in the process and stages of thrombus formation will require further investigation. In addition to its role in coagulation, polyP-induced Factor XII activation mediates the release of the inflammatory mediator bradykinin by activating the kallikrein-kinin system. This results in vascular leakage and edema formation in vivo. Together, polyP release from platelets has a physiological role in the thrombus-forming process, as well as in inflammatory reactions.     

Activation of bovine factor VII by hageman factor fragments

During the early events of coagulation of human blood by the intrinsic pathway, factor XII is activated to a form which can activate factor XI, and is proteolytically fragmented to smaller species (30,000 daltons and 70,000 daltons) which have lost most of the ability to activate factor XI but which can activate prekallikrein rapidly. The effect of these fragments on factor VII was studied. It was found that these Hageman factor fragments promoted rapid proteolysis of one-chain factor VII to a more active two-chain form. The amino-terminal sequences of the chains of activated factor VII were found to be Ala- Asx-Gly- and Ile-Val-Gly-, the same as were earlier observed after activation of factor VII by activated factor X. This finding indicates that initiation of coagulation by the intrinsic pathway also primes the extrinsic pathway.