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PubMed1986

Quantitative immunoblotting assay of blood coagulation factor XII.

The immunoblotting technique was applied to the study of Factor XII (F.XII) in plasma. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole plasma followed by electroblotting of the electropherograms to nitrocellulose (NC) membranes and immunologic detection by a double antibody technique was used. 125I-F.XII was transferred to the NC membrane in amounts proportional to the amount applied to the gel provided that a constant amount of carrier protein was present. Based on this, a quantitative assay was developed using either normal plasma or F.XII dilutions in F.XII-deficient plasma as standards. The measurement of F.XII antigen by immunoblotting was reproducible and gave values similar to those obtained by radial immunodiffusion. Two normal plasma pools contained 26 and 29 micrograms/ml of F.XII according to the immunoblotting assay. Compared to other immunoassays, immunoblotting has the advantage of directly estimating the apparent molecular weight (MW) of the protein of interest. Thus, we could confirm the normal apparent MW (80,000) of a F.XII-like molecule previously isolated from a cross reacting material (CRM)-positive F.XII-deficient plasma. None of eight CRM-negative F.XII-deficient plasmas showed an 80,000 MW immunoreactive molecule. However, five of these eight plasmas had a faint autoradiographic band at 115,000 MW that was similarly seen in only three out of 43 individual normal plasmas. The nature of this 115,000 MW band remains to be defined.

Autoradiography↗

PubMed1993

Human Hageman factor (factor XII) and high molecular weight kininogen compete for the same binding site on human umbilical vein endothelial cells.

Factor XII (FXII, plasma concentration 375 nM) is a critical member of the plasma contact activation system and is the zymogen form of FXIIa, a serine protease involved in intrinsic coagulation, complement activation, activation of factor VII, and generation of the vasoactive peptide bradykinin. As such its interaction with cells involved in inflammatory pathways can be of physiologic and pathologic significance. We have studied the binding of FXII to cultured human umbilical vein endothelial cells (HUVEC). HUVEC were incubated with 125I-FXII, and cell-bound factor FXII was measured. FXII bound to HUVEC saturably in a zinc-dependent manner. The optimal zinc concentration was 50-60 microM. Binding of labeled FXII was drastically reduced when a 200-fold molar excess of unlabeled FXII was included in the incubation mixture at time zero or when added at 60 min during a 150-min time course experiment. Quantitative binding experiments indicated a dissociation constant of 144 nM with 10-12 million binding sites/endothelial cell. Unlabeled high molecular weight kininogen (HK) inhibited the binding of labeled FXII with a Ki of 98 nM, whereas unlabeled FXII inhibited the binding of labeled HK to HUVEC with a Ki of 152 nM. SDS-polyacrylamide gel electrophoresis and autoradiography of cell-bound 125I-FXII showed that factor XII underwent limited proteolysis and the molecular weights of the fragments were similar in size to activated FXII. The cell-bound activated factor XII was also able to activate prekallikrein. These data suggest that (i) FXII binds to HUVEC specifically, saturably, and reversibly in a zinc-dependent manner, (ii) HK and FXII may compete with each other for the same cell-surface receptor/s, and (iii) cell-bound FXII is capable of undergoing activation to FXIIa.

Binding Sites↗

PubMed1984

Plasma prekallikrein, factor XII, antithrombin III, C1(-)-inhibitor and alpha 2-macroglobulin in critically ill patients with suspected disseminated intravascular coagulation (DIC).

In nine patients with suspected disseminated intravascular coagulation (DIC) and five controls, the following analyses were performed on admission and 7-29 hours later: Routine coagulation studies (fibrinogen, platelet count, fibrin(ogen) degradation products, ethanol gelation, reptilase time, Factor V) providing a semiquantitative DIC score, prekallikrein (PK), Factor XII, antithrombin III (AT-III), C1(-)-inhibitor and alpha 2-macroglobulin. Significant correlations were found: PK or AT III with the DIC-score, PK with AT-III and Factor XII, AT-III with Factor XII. The changes (expressed as a percentage of normal plasma) of PK and AT-III from the first to the second evaluation were nearly identical. The two patients with rapidly fatal irreversible shock showed the highest DIC score and a pronounced decrease of PK and AT-III, whereas in reversible shock stable or increasing PK and AT-III values were found. The other variables showed an overlap between reversible and irreversible shock. DIC in these shock patients, accompanied by a decrease in PK, probably was mediated via Factor XII activation. PK and AT-III might be of prognostic value in patients with (septic) shock.

Aged↗

PubMed1998

An automated chromogenic peptide substrate assay for coagulation factor XII.

We have developed an automated chromogenic peptide substrate assay for factor XII (FXIIcs) on a Cobas Mira S Plus clinical chemistry analyser using a new commercially available kit. This was used to determine factor XII (FXII) levels in plasma samples from 320 blood donors, 206 patients with a history of venous thrombosis and 74 lupus anticoagulant positive (LA+) patients. Results were compared with those obtained in a clotting assay for FXII (FXIIct) and an immunochemical assay (FXIIag). A satisfactory correlation coefficient of 0.92 and a regression line equation of y = 7.898 + 0.871x was obtained between FXIIcs and FXIIct in the 320 blood donors. Levels of FXII below the calculated normal range were found in nine blood donors (2.8%) and 16 venous thrombosis patients (7.8%). The blood donors and patients with venous thrombosis with low FXIIcs values had FXII levels below our lower limits of normal for both FXIIct and FXIIag; all were lupus anticoagulant negative. When FXII levels were determined in the 74 LA+ patients, 27 (36.5%) gave markedly lower FXII values in the FXIIct when compared with the FXIIcs. FXIIag levels corresponded with FXIIcs. The automated FXIIcs assay is therefore lupus anticoagulant insensitive and allows us to measure FXII levels accurately and routinely in large numbers of patient samples.

Autoanalysis↗

PubMed2006

Autoactivation of blood factor XII at hydrophilic and hydrophobic surfaces.

Contact activation of blood factor XII (FXII, Hageman factor) in neat-buffer solution is shown not to be specific for anionic hydrophilic procoagulants as proposed by the accepted biochemistry of surface activation. Rather, FXII activation in the presence of plasma proteins leads to an apparent specificity for hydrophilic surfaces that is actually due to a relative diminution of the FXII-->FXIIa reaction at hydrophobic surfaces. FXII activation in neat-buffer solution was effectively instantaneous upon contact with either hydrophilic (fully water-wettable clean glass) or hydrophobic (poorly water-wettable silanized glass) procoagulant particles, with greater FXIIa yield obtained by activation with hydrophobic procoagulants. In sharp contrast, both activation rate and yield was found to be significantly attenuated at hydrophobic surfaces in the presence of plasma proteins. Putative FXIIa produced by surface activation with both hydrophilic and hydrophobic procoagulants was shown to hydrolyze blood factor XI (FXI) to the activated form FXIa (FXIFXIIa-->FXIa) that causes FXI-deficient plasma to rapidly coagulate.

Biocompatible Materials↗

PubMed1987

Characterization of the human blood coagulation factor XII gene. Intron/exon gene organization and analysis of the 5'-flanking region.

A human genomic phage library was screened using a human factor XII cDNA as a hybridization probe. Two overlapping phage clones were isolated which contain the entire human factor XII gene. DNA sequence and restriction enzyme analysis of the clones indicate that the gene is approximately 12 kilobase pairs in size and is comprised of 13 introns and 14 exons. Exons 3-14 are contained in a genomic region of only 4.2 kilobase pairs with introns ranging in size from 80 to 554 base pairs. The coding sequence of factor XII consists of multiple putative domains that are homologous to putative domains found in fibronectin and tissue-type plasminogen activator. These regions were found as separate exons in the gene. The intron/exon gene organization is similar to the serine protease gene family of plasminogen activators and not to the clotting factor family. Analysis of the 5' end of the gene shows that it does not contain the typical TATA and CAAT sequences found in other genes. This is consistent with the finding that transcription of the gene is initiated at multiple sites.

Amino Acid Sequence↗

PubMed2005

Comparable levels of activity and antigen in factor XII deficiency: a study of 21 homozygotes and 58 heterozygotes.

Results of coagulation studies on 21 homozygote patients with factor XII (FXII) deficiency revealed that all of them had no cross-reacting material (CRM) in their plasma. The 58 heterozygotes had in every instance an antigen level comparable to that of clotting activity namely, approximately 50% of normal. An analysis of all pertinent literature also showed that the presence of CRM is very rare in FXII deficiency. CRM is present in approximately 5% of homozygote patients. More precisely, seven of 145 patients. Only in one case, the antigen level was normal (FXII Washington). This prevalence appears lower than that observed for another contact phase factor (prekallikrein). The significance of blood abnormal forms of FXII has not been completely clarified yet. Their study appears useful in the attempt of clarifying the structure-function relation of factor XII.

Blood Coagulation↗

PubMed1978

[Factor XII deficiency - Hageman trait. Report of 2 cases].

For the first time in our literature we have described the two cases of hereditary deficiency of factor XII (Hageman trait), a very rare disorder. It was discovered when a long clotting time was found in our patients during the course of a preoperative evaluation. Abnormal thrombelastogram, with a picture typical for hemophilia, gave as a reason to continue with laboratory investigation. The laboratory findings showed us a very low value of factor XII in one case, and total absence of factor XII in the other.

Adult↗

PubMed2001

Rapid and cooperative binding of factor XII to human umbilical vein endothelial cells.

When activated, factor XII (FXII) has been shown to play a role in a series of proteolytic cascades including systems as the fibrinolytic, the coagulation, the kallikrein-kinin and the complement. How FXII is activated in vivo remains poorly understood as the concentration and density of surface bound negative charges known to trigger the activation in vitro is far from sufficient in vivo. Specific binding of FXII to cellular receptors in the blood stream may, however, solve this problem which may be a question of inter molecular vicinity enhanced by binding to any surface. Here we report that the Zn(2+)-dependent binding of FXII to endothelial cells is rapid, saturable, specific and cooperative. Each endothelial cell from the human umbilical veins was found to bind (417 +/- 202) x 10(3) molecules of FXII with a Kd of (65 +/- 23) nM and a Hill coefficient of 2.1. The binding was inhibited by alpha-FXIIa but not by beta-FXIIa. The Kd for binding alpha-FXIIa was (50 +/- 27) nM. The rate of association was found to be 1.9 x 10(5) M(-1). min(-1). A confirmed inhibition by HK increased the Kd without affecting the maximal number of binding sites and the Hill coefficient. The concentration of HK in serum did not prevent binding of FXII/FXIIa to cells incubated with serum supplemented with Zn2+. The optimal concentration of Zn(2+) was 15 microM for binding factor XII/FXIIa whether purified or in serum.

Cells, Cultured↗

PubMed2005

Synergistic association between hypercholesterolemia and the C46T factor XII polymorphism for developing premature myocardial infarction.

Factor XII (FXII) plays a key role in both coagulation and fibrinolysis, thus its role in thrombotic processes is uncertain. Both genetic and environmental factors determine FXII plasma levels. A common C46T polymorphism in the Kozak region of F12 gene disturbs the translation of the protein leading to a significant reduction of FXII levels although its clinical significance is conflictive. We studied the F12 C46T polymorphism in 281 patients who had suffered from an acute myocardial infarction (MI) before 45-year-old and 550 control subjects from the same area. Serum levels of cholesterol, HDL, LDL, triglycerides and C reactive protein (CRP) were assayed in the MI group. The 46T allele slightly increased the risk to suffer from premature MI (OR: 1.64; 95% CI: 1.14-2.37; p = 0.008). Moreover, patients carrying the 46T allele showed increased levels of CRP (p = 0.002). Interestingly, we found that the simultaneous presence of the 46T allele and hypercholesterolemia increases the risk to develop premature MI 2.26 times. The F12 C46T polymorphism, associated with a reduction of plasma FXII levels, seems to play a deleterious effect, predisposing the development of premature MI, especially in hypercholesterolemic patients. This effect could be associated with an increased pro-inflammatory state, as the 46T allele associates with high levels of CRP.

Age Factors↗

PubMed1985

Effect of heparin on the inactivation rate of human activated factor XII by antithrombin III.

Human antithrombin III (ATIII) is a plasma inhibitor of several serine proteases of the blood coagulation system. Previous investigations have reported that the presence of heparin has a multifold accelerating effect on the inhibition of factor XIIa and XIIf, the active species derived from factor XII. Recent studies from our laboratories have confirmed that ATIII inactivates factor XIIa and factor XIIf, but only contributes 2% to 3% to the inhibition of activated factor XII species in plasma. The major inhibitor is C1 inhibitor. Therefore, we have reexamined the heparin effect on the rate of inhibition of factor XIIa and factor XIIf in purified systems. We also have studied the effect of heparin on the inactivation of both factor XII-derived active species by various plasmas. Using purified factor XIIa and ATIII, we found that heparin (0.7 to 34.0 U/mL) increased the rate of inhibition of Factor XIIa. However, at heparin concentrations usually achieved during anticoagulant therapy (0.7 U/mL), the inhibition was accelerated only fourfold. This implies only a 6% contribution to the inhibitory effect of plasma. This suggestion was confirmed by the observation that heparin (1.5 U/mL) added to factor XII-deficient plasma and reconstituted with factor XIIa did not produce a detectable enhancement of the rate of inhibition of factor XIIa. Furthermore, using purified factor XIIf and antithrombin III, heparin (3.6 to 57.2 U/mL) increased the inactivation rate constant of factor XIIf by 1.6 to 14.0 times. This small effect was confirmed by the observation that heparin at a concentration greater than that sufficient for anticoagulation (1.4 U/mL) did not modify the inactivation rate of factor XIIf by prekallikrein-deficient plasma, and thus C1 inhibitor remains the major inhibitor even in the presence of heparin. From this study and our previous investigations on the effect of heparin on the inhibition of kallikrein and factor XIa, we conclude that heparin does not significantly affect the protease activity of purified contact activation factors or the activities expressed by these proteases in plasma.

Antithrombin III↗

PubMed1983

Effect of DDAVP on plasma level of factor XII.

The effect of DDAVP on blood coagulation factors was investigated after its intravenous infusion into normal subjects. A marked increase in factor XII was observed in addition to the expected rise of factor VIII coagulant activity (VIII:C), factor VIII related antigen (VIIIR:Ag) and plasminogen activator, DDAVP also produced a concomitant but less pronounced rise of factor VII, but there was no change in factors V, IX, X and XI.

Adult↗

PubMed2003

Porcine endometrial expression of kininogen, factor XII, and plasma kallikrein in cyclic and pregnant gilts.

Establishment of pregnancy in the pig is accompanied by a localized uterine acute inflammatory response and increase in uterine blood flow. Following rapid trophoblast elongation on Day 12 of pregnancy there is an increase in tissue kallikrein activity and release of bradykinin into the uterine lumen, suggesting the kallikrein-kininogen-kinin system is active in the porcine uterus. The present study investigated endometrial expression and presence of the various factors of the kallikrein-kininogen-kinin system. Endometrial L- and H-kininogen gene expression as well as presence of kininogens in the uterine flushings was evaluated throughout the estrous cycle and early pregnancy in the pig. The possible involvement of plasma kallikrein and Factor XII, activators of the kallikrein-kininogen-kinin system, were evaluated through analysis of gene expression in endometrial and conceptus tissues. Gene expression for plasma kallikrein, Factor XII, and H-kininogen were detected in endometrium but not early conceptus tissues. Factor XII and H-kininogen gene expression were similar across the days of the estrous cycle and early pregnancy. Endometrial plasma kallikrein gene expression was low but increased on Day 15 of the estrous cycle, whereas expression was similar across the days of early pregnancy. In comparison to cyclic gilts, endometrial L-kininogen gene expression increased fourfold on Days 15 and 18 of pregnancy. Both L- and H-kininogen were detected in the uterine flushings of cyclic and pregnant gilts. Presence of L- and H-kininogen in the porcine uterus and endometrial gene expression of plasma kallikrein and Factor XII provide evidence that the kallikrein-kininogen-kinin system is biologically active during establishment of pregnancy in the pig.

Animals↗

PubMed2006

Probing single-stranded DNA and its biomolecular interactions through direct catalytic activation of factor XII, a protease of the blood coagulation cascade.

Triggered self-activation of factor XII, a blood coagulation protease, was utilized for the amplified visual detection of ss-DNA targets in a non-sequence specific way. Factor XII holds potential as a low-affinity and therefore non-interfering probe for DNA secondary structure and for the screening of protein binding to ss-DNA. The observation that ss-DNA also accelerates coagulation of human blood plasma is relevant to the emerging field of aptamer therapeutics.

Blood Coagulation↗

PubMed1996

Identification of the zinc-dependent endothelial cell binding protein for high molecular weight kininogen and factor XII: identity with the receptor that binds to the globular "heads" of C1q (gC1q-R).

High molecular weight kininogen (HK) and factor XII are known to bind to human umbilical vein endothelial cells (HUVEC) in a zinc-dependent and saturable manner indicating that HUVEC express specific binding site(s) for those proteins. However, identification and immunochemical characterization of the putative receptor site(s) has not been previously accomplished. In this report, we have identified a cell surface glycoprotein that is a likely candidate for the HK binding site on HUVECs. When solubilized HUVEC membranes were subjected to an HK-affinity column in the presence or absence of 50 microM ZnCl2 and the bound membrane proteins eluted, a single major protein peak was obtained only in the presence of zinc. SDS/PAGE analysis and silver staining of the protein peak revealed this protein to be 33 kDa and partial sequence analysis matched the NH2 terminus of gC1q-R, a membrane glycoprotein that binds to the globular "heads" of C1q. Two other minor proteins of approximately 70 kDa and 45 kDa were also obtained. Upon analysis by Western blotting, the 33-kDa band was found to react with several monoclonal antibodies (mAbs) recognizing different epitopes on gC1q-R. Ligand and dot blot analyses revealed zinc-dependent binding of biotinylated HK as well as biotinylated factor XII to the isolated 33-kDa HUVEC molecule as well as recombinant gC1q-R. In addition, binding of 125I-HK to HUVEC cells was inhibited by selected monoclonal anti-gC1q-R antibodies. C1q, however, did not inhibit 125I-HK binding to HUVEC nor did those monoclonals known to inhibit C1q binding to gC1q-R. Taken together, the data suggest that HK (and factor XII) bind to HUVECs via a 33-kDa cell surface glycoprotein that appears to be identical to gC1q-R but interact with a site on gC1q-R distinct from that which binds C1q.

Antibodies, Monoclonal↗

PubMed1985

A monoclonal antibody that inhibits activation of human Hageman factor (factor XII).

A monoclonal antibody to human Hageman factor (HF, factor XII) was derived from BALB/c mouse spleen cells fused with NS-1 mouse myeloma cells. This antibody, purified from ascites fluid, reacted with HF to inhibit the activation of HF, purified or in normal pooled plasma, as measured by a coagulation assay. The antibody did not inhibit the coagulant activity of activated HF. The antibody also inhibited the generation of amidolytic activity in HF-ellagic acid mixtures, but failed to inhibit the amidolytic properties of the carboxy-terminal fragment of HF (HFf). Amidolytic activity, absent in an HF-monoclonal antibody mixture, was generated upon treatment with insoluble trypsin. Monoclonal antibody, bound to CNBr Sepharose 4B gel (Pharmacia Fine Chemicals, Piscataway, NJ), reversibly bound HF in plasma or in buffer, without activating it. HF was then eluted with 4 mol/L guanidine HCI. The passage of 125I-labeled HF enzymatically cleaved by trypsin through a column of monoclonal antibody-CNBr Sepharose 4B gel resulted in flow-through of HFf with a molecular weight (mol wt) of 30,000 and HF fragments of mol wt 12,000. Elution with 4 mol/L guanidine HCI yielded several HF fragments (mol wt 80,000, 52,000, and 40,000) but not HFf. These data suggest that the single determinant recognized by the murine monoclonal antibody is not on HFf, but rather on the amino-terminal fragment thought to be involved in the binding activity of HF. The monoclonal anti-HF bound to CNBr-activated Sepharose 4B gel could be used to artificially deplete plasma samples of HF.

Animals↗

PubMedDate not supplied

Pretransplant factor XII levels correlate with prognosis in patients undergoing autologous graft for hematological malignancies.

A retrospective analysis of data collected in a previous study suggested that pre-conditioning levels of factor XII might have prognostic value in autologous graft recipients. In order to confirm whether pre-transplant factor XII (pFXII) levels could be correlated with outcome, seventy-six (35 autologous and 41 allogeneic) transplant recipients were prospectively evaluated. A significant direct relationship was found between pFXII levels and both overall and disease-free survival in the autologous grafts, but not in the allogeneic ones. Although the molecular mechanisms of this relationship still need to be clarified, these data seem to justify larger efforts to confirm whether factor XII (FXII) assay should be used in pre-transplant evaluation of patients.

Adolescent↗

PubMed1994

Dextran sulphate inhibits phospholipid and sulphatide mediated autoactivation of factor XII.

In normal plasma, high molecular mass dextran sulphate (DS500) induces formation of amidolytic activity towards the chromogenic substrate H-D-Pro-Phe-Arg-p-nitroanilide (S-2302) specific to factor XII and kallikrein. No amidolytic activity was formed when plasma deficient in prekallikrein was exposed to DS500. In contrast, factor XII amidolytic activity was formed upon exposure to sulphatide or acidic phospholipids. To assess whether DS500 interferes with the sulphatide and the acidic phospholipid in activating factor XII, plasma deficient in prekallikrein was incubated with phosphatidylinositol phosphate (PtdInsP) and sulphatide at the conditions necessary for activation with these surfaces and various concentrations of DS500. DS500 inhibited both the PtdInsP and the sulphatide-mediated autoactivation in an antithrombin III independent manner. Heparin also inhibited the PtdInsP mediated autoactivation but not that mediated by sulphatide. The heparin inhibition was due to enhancement of the antithrombin III activity, which could be partly blocked by preincubation of plasma with rabbit anti-human antithrombin III IgG.

Amino Acid Sequence↗