[New perspective of anticoagulation in intensive care unit: basic and clinical advances in coagulation factor XII and XI inhibitors].

Anticoagulation therapy stands as a key treatment for thrombotic diseases. The consequential bleeding risk tied to existing anticoagulation methods significantly impacts patient prognosis. In the intensive care unit (ICU), patients often necessitate organ support, leading to the inevitable placement of artificial devices in blood vessels, thereby requiring anticoagulation treatment to avert clot formation that might impede organ support. Nevertheless, these patients commonly encounter a heightened risk of bleeding. Hemophilia B, identified in 1953, manifests as a deficiency in coagulation factor XI (FXI), which focused people's perspective on the endogenous coagulation pathway, that is, the contact pathway. Upon interaction between the surface of artificial devices and FXII, FXII activates, subsequently triggering FXI and initiating the "coagulation cascade" within the contact pathway. Inhibitors targeting the contact pathway encompass two primary categories: FXII inhibitors and FXI inhibitors, capable of impeding this process. This article reviews the role of FXII and FXI in activating the contact pathway, seeking to illuminate their contributions to thrombus formation. By listing the relatively mature drugs and their indications, clinicians are familiar with this new anticoagulant.

Tackling the Root Cause of Surface-Induced Coagulation: Inhibition of FXII Activation to Mitigate Coagulation Propagation and Prevent Clotting.

Factor XII (FXII) is a zymogen present in blood that tends to adsorb onto the surfaces of blood-contacting medical devices. Once adsorbed, it becomes activated, initiating a cascade of enzymatic reactions that lead to surface-induced coagulation. This process is characterized by multiple redundancies, making it extremely challenging to prevent clot formation and preserve the properties of the surface. In this study, a novel modulatory coating system based on C1-esterase inhibitor (C1INH) functionalized polymer brushes, which effectively regulates the activation of FXII is proposed. Using surface plasmon resonance it is demonstrated that this coating system effectively repels blood plasma proteins, including FXII, while exhibiting high activity against activated FXII and plasma kallikrein under physiological conditions. This unique property enables the modulation of FXII activation without interfering with the overall hemostasis process. Furthermore, through dynamic Chandler loop studies, it is shown that this coating significantly improves the hemocompatibility of polymeric surfaces commonly used in medical devices. By addressing the root cause of contact activation, the synergistic interplay between the antifouling polymer brushes and the modulatory C1INH is expected to lay the foundation to enhance the hemocompatibility of medical device surfaces.

Coming soon to a pharmacy near you? FXI and FXII inhibitors to prevent or treat thromboembolism.

Anticoagulants have been in use for nearly a century for the treatment and prevention of venous and arterial thromboembolic disorders. The most dreaded complication of anticoagulant treatment is the occurrence of bleeding, which may be serious and even life-threatening. All available anticoagulants, which target either multiple coagulation factors or individual components of the tissue factor (TF) factor VIIa or the common pathways, have the potential to affect hemostasis and thus to increase bleeding risk in treated patients. While direct oral anticoagulants introduced an improvement in care for eligible patients in terms of safety, efficacy, and convenience of treatment, there remain unmet clinical needs for patients requiring anticoagulant drugs. Anticoagulant therapy is sometimes avoided for fear of hemorrhagic complications, and other patients are undertreated due to comorbidities and the perception of increased bleeding risk. Evidence suggests that the contact pathway of coagulation has a limited role in initiating physiologic in vivo coagulation and that it contributes to thrombosis more than it does to hemostasis. Because inhibition of the contact pathway is less likely to promote bleeding, it is an attractive target for the development of anticoagulants with improved safety. Preclinical and early clinical data indicate that novel agents that selectively target factor XI or factor XII can reduce venous and arterial thrombosis without an increase in bleeding complications.

An Update on Safe Anticoagulation.

Blood coagulation is essential to maintain the integrity of a closed circulatory system (hemostasis), but also contributes to thromboembolic occlusion of vessels (thrombosis). Thrombosis may cause deep vein thrombosis, pulmonary embolism, myocardial infarction, peripheral artery disease, and ischemic stroke, collectively the most common causes of death and disability in the developed world. Treatment for the prevention of thromboembolic diseases using anticoagulants such as heparin, coumarins, thrombin inhibitors, or antiplatelet drugs increase the risk of bleeding and are associated with an increase in potentially life-threatening hemorrhage, partially offsetting the benefits of reduced coagulation. Thus, drug development aiming at novel targets is needed to provide efficient and safe anticoagulation. Within the last decade, experimental and preclinical data have shown that some coagulation mechanisms principally differ in thrombosis and hemostasis. The plasma contact system protein factors XII and XI, high-molecular-weight kininogen, and plasma kallikrein specifically contribute to thrombosis, however, have minor, if any, role in hemostatic coagulation mechanisms. Inherited deficiency in contact system proteins is not associated with increased bleeding in humans and animal models. Therefore, targeting contact system proteins provides the exciting opportunity to interfere specifically with thromboembolic diseases without increasing the bleeding risk. Recent studies that investigated pharmacologic inhibition of contact system proteins have shown that this approach provides efficient and safe thrombo-protection that in contrast to classical anticoagulants is not associated with increased bleeding risk. This review summarizes therapeutic and conceptual developments for selective interference with pathological thrombus formation, while sparing physiologic hemostasis, that enables safe anticoagulation treatment.

Differing mechanisms of thrombin generation in live haematological and solid cancer cells determined by calibrated automated thrombography.

: Calibrated automated thrombography (CAT) is emerging as a reliable tool for real-time estimation of thrombin generation potential. There is a clinical need for knowledge about the pathways underlying the thrombotic phenotype of different malignancies. Cells from solid (e.g. pancreatic cancer; n = 7) and malignant haematological cell lines (e.g. multiple myeloma; n = 5) were evaluated for thrombin generation, using CAT, with the addition of control plasma (NormTrol; Helena Biosciences, Gateshead, UK)) or plasma deficient in coagulation factors VII and XII. In addition, tissue factor (TF) cell surface expression was determined by flow cytometry. In platelet-free plasma, thrombin generation in all cancer cell lines was cell concentration dependent, with the pancreatic cancer line CFPAC-1 producing the highest thrombin of 220 nmol/l at 5 × 10-cells/ml concentration. Lag times and times to peak reflected most significant differences out of all thrombin generation parameters measured and were inversely correlated with cell surface TF surface expression. Solid tumour cell lines had higher thrombin peaks, faster lag times, and a thrombin generation profile of overall greater magnitude than haematological cell lines. In the absence of factor VII in platelet-free plasma, thrombin generation in solid pancreatic cancer cell lines was significantly reduced unlike in haematological cell lines. However, in the absence of factor XII, thrombin generation was reduced more in haematological cells but had little or no effect on solid cell lines. The CAT assay identified characteristic differences in thrombin generation kinetics between solid tumour and haematological cancer cell lines, of which lag time and time to peak correlated with TF cell surface expression.

FXII promotes proteolytic processing of the LRP1 ectodomain.

BACKGROUND: Factor XII (FXII) is a serine protease that is involved in activation of the intrinsic blood coagulation, the kallikrein-kinin system and the complement cascade. Although the binding of FXII to the cell surface has been demonstrated, the consequence of this event for proteolytic processing of membrane-anchored proteins has never been described. METHODS: The effect of FXII on the proteolytic processing of the low-density lipoprotein receptor-related protein 1 (LRP1) ectodomain was tested in human primary lung fibroblasts (hLF), alveolar macrophages (hAM) and in human precision cut lung slices (hPCLS). The identity of generated LRP1 fragments was confirmed by MALDI-TOF-MS. Activity of FXII and gelatinases was measured by S-2302 hydrolysis and zymography, respectively. RESULTS: Here, we demonstrate a new function of FXII, namely its ability to process LRP1 extracellular domain. Incubation of hLF, hAM, or hPCLS with FXII resulted in the accumulation of LRP1 ectodomain fragments in conditioned media. This effect was independent of metalloproteases and required FXII proteolytic activity. Binding of FXII to hLF surface induced its conversion to FXIIa and protected FXIIa against inactivation by a broad spectrum of serine protease inhibitors. Preincubation of hLF with collagenase I impaired FXII activation and, in consequence, LRP1 cleavage. FXII-triggered LRP1 processing was associated with the accumulation of gelatinases (MMP-2 and MMP-9) in conditioned media. CONCLUSIONS: FXII controls LRP1 levels and function at the plasma membrane by modulating processing of its ectodomain. GENERAL SIGNIFICANCE: FXII-dependent proteolytic processing of LRP1 may exacerbate extracellular proteolysis and thus promote pathological tissue remodeling.

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time.

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

Factor XII stimulates ERK1/2 and Akt through uPAR, integrins, and the EGFR to initiate angiogenesis.

Factor XII (FXII) and high molecular weight kininogen (HK) mutually block each other's binding to the urokinase plasminogen activator receptor (uPAR). We investigated if FXII stimulates cells by interacting with uPAR. FXII (3-62nM) with 0.05mM Zn(2+) induces extracellular signal-related kinase 1/2 (ERK1/2; mitogen-activated protein kinase 44 [MAPK44] and MAPK42) and Akt (Ser473) phosphorylation in endothelial cells. FXII-induced phosphorylation of ERK1/2 or Akt is a zymogen activity, not an enzymatic event. ERK1/2 or Akt phosphorylation is blocked upstream by PD98059 or Wortmannin or LY294002, respectively. An uPAR signaling region for FXII is on domain 2 adjacent to uPAR's integrin binding site. Cleaved HK or peptides from HK's domain 5 blocks FXII-induced ERK1/2 and Akt phosphorylation. A beta(1) integrin peptide that binds uPAR, antibody 6S6 to beta(1) integrin, or the epidermal growth factor receptor (EGFR) inhibitor AG1478 blocks FXII-induced phosphorylation of ERK1/2 and Akt. FXII induces endothelial cell proliferation and 5-bromo-2'deoxy-uridine incorporation. FXII stimulates aortic sprouting in normal but not uPAR-deficient mouse aorta. FXII produces angiogenesis in matrigel plugs in normal but not uPAR-deficient mice. FXII knockout mice have reduced constitutive and wound-induced blood vessel number. In sum, FXII initiates signaling mediated by uPAR, beta(1) integrin, and the EGFR to induce human umbilical vein endothelial cell proliferation, growth, and angiogenesis.

The interaction of coagulation factor XII and monocyte/macrophages mediating peritoneal metastasis of epithelial ovarian cancer.

OBJECTIVES: Pathological studies have indicated that the peritoneum of epithelial ovarian cancer (EOC) patients exhibits characteristics of chronic inflammation like peritonitis. Abundant macrophage infiltration and increased expression of coagulation factor XII (FXII) have been observed in the peritoneum of EOC patients. The aim of this study is to determine how the interaction between FXII and monocyte/macrophages (MO/MAs) contributes to EOC cell invasion and metastasis of the peritoneum. METHODS: MO/MAs from the peripheral blood of healthy female donors and tumor-associated macrophages (TAMs) from EOC ascites were collected and cultured. We assessed phenotypes, cytokine/chemokine production, and phagocytic function of FXII-treated MO/MAs. The effects of the FXII-MO/MAs interaction on EOC cell invasion were determined by the Matrigel in vitro invasion assay. In addition, signaling pathway mediators were evaluated for their potential roles in MO/MA activation. RESULTS: MO/MAs exhibited M2-polarized phenotypes after FXII treatment, which was CD163(high)IL-10(high)CCL18(high)IL-8(high)CCR2(high)CXCR2(high). The phagocytic potential of MO/MAs was also upregulated. Matrigel results indicated that invasion of EOC cells was enhanced when exposed to conditioned medium from FXII-stimulated MO/MAs. Transcription factors found to be upregulated in FXII-stimulated MO/MAs included Fra-1, Fra-2, Fos-B in the AP-1 family, oncogenes HIF-1 and Oct, and STAT-5A in the STAT family. CONCLUSIONS: FXII may facilitate EOC cell metastasis by transforming MO/MAs toward tumor-associated macrophage-like cells.

Hemostyptic effect of oxidized cellulose on blood platelets.

Cellulose is one of the hemostyptic biomaterials, which are able to initiate or accelerate blood coagulation at the site of their application. It belongs to surgical sealants. The mechanism of its action is not clearly understood. We studied the participation of blood platelets in this mechanism. As a marker of platelet activation we used serotonin release reaction. Serotonin release in platelet rich plasma incubated with various concentrations of oxidized cellulose (0.5%-2.0%) started in about 20 min. Washed platelets were not directly activated by oxidized cellulose within one hour. Washed platelets reconstituted in plasma obtained from two patients with coagulation factor XII deficiency were activated by oxidized cellulose with a prolonged lag phase. Our results demonstrate the significant influence of factor XII on blood platelets activation by oxidized cellulose.

Binding of activated Factor XII to endothelial cells affects its inactivation by the C1-esterase inhibitor.

It is well known that activated Factor XII (FXIIa) and kallikrein are rapidly inactivated in plasma as a result of reaction with endogenous inhibitors. The purpose of this may be to prevent uncontrolled deleterious spreading and activation of target zymogens. Both FXII and the complex plasma prekallikrein/high molecular mass kininogen become activated when they bind, in a Zn2+-dependent manner, to receptors on human umbilical vein endothelial cells (HUVEC). The C1-esterase inhibitor (C1-INH) is by far the most efficient inhibitor of FXIIa. In the present study it has been investigated whether binding of FXIIa to HUVEC might offer protection against inactivation by C1-INH. It appeared that the relative amidolytic activity of purified FXIIa bound to the surface of HUVEC decreased according to the concentration of C1-INH in medium; however, the decrease was smaller than that measured for inactivation of FXIIa in solution. The secondary rate constant for the inactivation was 3-10-fold lower for cell-bound than for soluble FXIIa. The inactivation was found to be caused by C1-INH binding to cell-bound FXIIa. Accordingly, the amidolytic activity of saturated amounts of cell-bound FXIIa was reduced in the presence of C1-INH and was theoretically nonexistent at physiological C1-INH concentrations. Amidolytic activity was, however, present on HUVEC incubated with plasma indicating that the endogenous C1-INH did not completely abolish the activity of FXIIa generated during the incubation period. This supports the hypothesis that binding to endothelial cells protects the activated FXII against inactivation by its major endogenous inhibitor. Hence, the function of FXII may be localized at cellular surfaces.

The sympathoadrenal system mediates the blood pressure and cardiac effects of human coagulation factor XII-related "new pressor protein".

OBJECTIVE: To investigate the major cardiovascular effects of human plasma "new pressor protein" (NPP) and how the adrenal medulla contributes to these effects. METHODS: NPP was injected into bioassay rats intravenously, and the effects on blood pressure and cardiac function were investigated. Acute adrenal medullectomy (2MDX), alpha- and beta-adrenergic blockade and plasma catecholamine levels were also used to evaluate the role of the sympathoadrenal system in mediating the NPP effects. RESULTS: NPP significantly raised systolic blood pressure (SBP) and mean arterial pressure but not diastolic blood pressure (DBP), with no significant change in total peripheral resistance. Heart rate, cardiac output and stroke volume rose by 16%, 53% and 36%, respectively. Plasma catecholamines increased massively, notably adrenaline, raising the adrenaline to noradrenaline ratio from about 4:1 to 18:1. 2MDX attenuated the increments of SBP and heart rate by more than 90% and more than 70%, respectively, implicating the adrenal medulla. Beta-adrenergic blockade (propranolol) potentiated the NPP-induced increase of SBP and DBP, but not that of heart rate. Combined alpha- and beta-adrenergic blockade (phentolamine and propranolol) blocked the rise in SBP, DBP and heart rate. CONCLUSIONS: NPP's hypertensive action is attributable mainly to increases in systolic blood pressure, heart rate and cardiac output (an increase in heart rate and stroke volume) with massive release of adrenal medullary catecholamines. Such effects suggest a novel axis between coagulation factor XII and the sympathoadrenal system, the cardiovascular effects of which are controlled by combined alpha- and beta-adrenergic blockade, but not by angiotensin-converting enzyme inhibition. Clinical relevance depends on whether NPP is formed in vivo in thrombotic states.

Human coagulation factor XII-related "new pressor protein": role of PACAP in its cardiovascular and sympathoadrenal effects.

OBJECTIVE: To investigate the role of pituitary adenylate cyclase-activating polypeptide (PACAP)-38 and -27 as possible mediators of the actions of "new pressor protein" (NPP), which is related to coagulation factor XIIa, on blood pressure (systolic and diastolic blood pressure) and heart rate, and on the release of adrenal medullary catecholamines. METHODS: Adult male Wistar bioassay rats (n=8 to 18 per group) were anesthetized with inactin (100 mg/kg intraperitoneally), ganglion-blocked with pentolinium (19.2 mg/kg subcutaneously) and treated with captopril (2.5 mg/kg intravenously). Human NPP was injected at 20 L plasma equivalent per approximately 300 g of rat intravenously, and both PACAPs were injected at 10 g/kg intravenously. The systolic and diastolic blood pressure and heart rate responses to all of these agonists were determined using a MacLab/8 system. Arterial plasma adrenaline and noradrenaline were determined by high performance liquid chromatography with fluorimetric detection. Responses to NPP, the PACAPs and a specific PACAP antagonist were compared to assess the PACAPs as potential mediators of the cardiovascular effects of NPP. RESULTS: PACAP-38 mimicked the effects of NPP on systolic and diastolic blood pressure and heart rate more closely than did PACAP-27. Generally, NPP raised diastolic blood pressure and heart rate, and especially plasma adrenaline and noradrenaline, more impressively in degree and duration than that achieved by the PACAPs. The antagonism of PACAP receptors (PAC-1) significantly reduced the cardiovascular effects of NPP by 30% to 50%. CONCLUSIONS: PACAP-38, especially, may qualify as a potential mediator of the cardiovascular and sympathoadrenal effects of NPP but incomplete inhibition of NPP activity by PAC-1 receptor antagonism and the observed differences all suggest that PACAP is not the only peptide involved. Such peptidic mediation of the effects of NPP may explain the potentiation of NPP by captopril and why NPP remains effective after cholinergic blockade. These data suggest that PACAP is involved in a novel axis between NPP, cardiac function and blood pressure that resists angiotensin-converting enzyme inhibition. Any endogenous production of NPP could raise clinically relevant issues pertaining to therapy with ACE inhibitors.

Tryptase's potent mitogenic effects in human airway smooth muscle cells are via nonproteolytic actions.

We reported previously that mast cell tryptase is a growth factor for dog tracheal smooth muscle cells. The goals of our current experiments were to determine if tryptase also is mitogenic in cultured human airway smooth muscle cells, to compare its strength as a growth factor with that of other mitogenic serine proteases, and to determine whether its proteolytic actions are required for mitogenesis. Highly purified preparations of human lung beta-tryptase (1-30 nM) caused dose-dependent increases in DNA synthesis in human airway smooth muscle cells. Maximum tryptase-induced increases in DNA synthesis far exceeded those occurring in response to coagulation cascade proteases, such as thrombin, factor Xa, or factor XII, or to other mast cell proteases, such as chymase or mastin. Irreversibly abolishing tryptase's catalytic activity did not alter its effects on increases in DNA synthesis. We conclude that beta-tryptase is a potent mitogenic serine protease in cultured human airway smooth muscle cells. However, its growth stimulatory effects in these cells occur predominantly via nonproteolytic actions.

Factor XI assembly and activation on human umbilical vein endothelial cells in culture.

Biotin-FXI optimally bound to HUVEC in the presence of 40 nM high molecular weight kininogen (HK) and > or =7 microM Zn2+. There was little specific FXI binding in the absence of added HK and at concentrations of Zn2+ <15 microM. FXI and prekallikrein, but not prothrombin, blocked biotin-FXI binding to HUVEC in the presence of HK with an IC50 of 18 nM and 180 nM, respectively. Monoclonal antibody HKL16 and peptide SDD31 also inhibited biotin-XI binding in the presence of HK with an IC50 of 4.7 nM and 50 microM, respectively. Alternatively, peptide T249-F260 of FXI's apple domain 3 and heparin monosulfate were weak inhibitors of FXI binding to HUVEC. FXI bound to HUVEC with an apparent Kd of 6.9 +/- 3.0 nM and Bmax of 13 +/- 2.6 x 10(6) sites/cell. FXI bound to HK on HUVEC, but not prothrombin, became converted to FXIa. FXI activation on HUVEC resulted from tissue culture media bovine factor XIIa. HUVEC grown in human factor XI-deficient serum did not support FXI activation. FXI binding to HUVEC in culture was mostly mediated by HK and FXI activation on HUVEC is dependent on cell-associated factor XIIa.

Activation of the kinin-forming cascade on the surface of endothelial cells.

Activation of the plasma kallikrein-kinin forming cascade takes place upon incubation with human umbilical vein endothelial cells. The mechanism by which initiation occurs is uncertain. Zinc-dependent binding of plasma proteins to gC1qR, cytokeratin 1, and perhaps u-PAR is requisite for activation to take place. We demonstrate here that during a 2 hour incubation time plasma deficient in either factor XII or high molecular weight kininogen (HK) fails to activate, as compared to normal plasma, but with more prolonged incubation, factor XII-deficient plasma gradually activates while HK-deficient plasma does not. Our data support both factor XII-dependent (rapid) and factor XII-independent (slow) mechanisms; the latter may require a cell-derived protease to activate prekallikrein and the presence of zinc ions and HK.

Factor XII-dependent contact activation on endothelial cells and binding proteins gC1qR and cytokeratin 1.

Although proteins of the kinin-forming pathway are bound along the surface of endothelial cells, the mechanism of activation of this proteolytic cascade is unclear. Endothelial cell surface proteins, gC1qR and cytokeratin 1, are capable of binding Factor XII and high molecular weight kininogen (HK) in a zinc-dependent reaction thus we considered the possibility that these proteins might catalyze initiation of the cascade. Incubation of Factor XII, prekallikrein, and HK with gC1qR or cytokeratin 1 leads to a zinc-dependent and Factor XII-dependent conversion of prekallikrein to kallikrein. We also demonstrate that normal plasma is capable of activating upon interaction with the cells whereas plasma deficient in Factor XII, prekallikrein and HK do not activate. Normal plasma activation was inhibitable by antibody to gC1qR and cytokeratin 1. Thus, gC1qR and cytokeratin 1, represent potential initiating surfaces for activation of the plasma kinin-forming cascade and may do so as a result of their expression along cell surfaces.

The region 1-11 of Alzheimer amyloid-beta is critical for activation of contact-kinin system.

Amyloid-beta protein (Abeta) has been implicated in the pathogenesis of Alzheimer's disease (AD) because of its neurotoxicity and its ability to trigger a local inflammatory response. In the present study using truncated Abeta peptides, we identified the region between residues 1 and 11 as critical for the activation of the contact system in vitro through an ionic interaction of Abeta with factor XII and/or kallikrein. Concomitant incubation of a small cationic peptide (lysine(4)) with Abeta abrogated its ability to trigger the cleavage of high molecular weight kininogen, indicating that Abeta's activity can be blocked by an inhibitory peptide. These findings could be clinically important, since there is evidence that the contact system is activated in AD brain. Thus, prevention of contact system activation, beside diminishing the recruitment of glial cells and microvascular permeability, can also decrease the activation of complement system and the release of IL6, both factors being considered to play an important role in the inflammatory reactions in AD brain.

Factor XII does not initiate prekallikrein activation on endothelial cells.

It is well known that on artificial surfaces, binding and autoactivation of factor XII (FXII) is the initiating event of plasma prekallikrein (PK) activation. We performed investigations to examine whether this mechanism was true for FXII activation on endothelial cells (HUVEC). Activation of PK on HUVEC required an optimal substrate and Zn2+ concentration, the latter of which varied with the buffer's carrier protein. Maximal PK activation required the addition of 250 microM or 10 microM Zn2+ to buffers containing bovine serum albumin (BSA) or gelatin, respectively. However, the actual free Zn2+ concentration in these buffers was the same at 8 microM. In both BSA- and gelatin-containing buffers and using two different chromogenic substrates for FXII, no autoactivation of FXII on HUVEC was seen when incubated for up to 60 min. Rather, initiation of FXII enzymatic activity required the presence of PK. FXII activation after PK activation contributed to the extent of measured enzymatic activity, but its role was secondary because treatment with corn trypsin inhibitor or a neutralizing antibody to FXIIa did not abolish the measured enzymatic activity. They also reduced the activity to the level seen with PK activation alone. Alternatively, soybean trypsin inhibitor abolished the proteolytic activity associated with PK and FXII activation on HUVEC. Further, only normal human and FXII-deficient plasmas, not PK-deficient plasma, had the ability to generate proteolytic activity when incubated over endothelial cells. In a purified system, maximal PK activation was measured after a 10-15 min incubation depending upon the concentration of reactants. When FXII was added with the PK, maximal activation occurred within 7.5-10 min. In normal human or FXII-deficient plasmas, but not in PK-deficient plasma, maximal activation was seen in 4 min. These data indicate that on HUVEC, unlike artificial surfaces, PK activation when bound to HK is the initiating activation event in this system. FXII activation is secondary to PK activation and contributes to the extent of measured enzymatic activity. These data challenge the accepted dogmas of "contact activation" and suggest that on biologic membranes a new notion as to how this system is activated needs to be considered.

Sulfatide can markedly enhance thrombogenesis in rat deep vein thrombosis model.

Although sulfatide (galactosylceramide I3-sulfate) has been reported to activate blood coagulation factor XII (Hageman factor), it has been administered to animals without subsequent thrombus formation. We recently found that sulfatide binds to fibrinogen and thus disturbs fibrin formation in vitro, suggesting its possible role as an anticoagulant rather than as a coagulant. We therefore examined the in vivo effects of sulfatide on thrombogenesis by using a rat deep vein thrombosis model in which thrombus is induced by ligating the inferior vena cava. Sulfatide and gangliosides were each separately administered to rats 1 min before the vein ligation, and after 3 h, sulfatide but not gangliosides markedly (P < .001) enhanced the thrombogenesis. A kinetic turbidmetric assay of plasma coagulation initiated by CaCl2 in the wells of a microtiter plate revealed that coagulation was also markedly accelerated in the presence of sulfatide but not gangliosides, the results of which seemed to be very consistent with those of the in vivo experiments. Because sulfatide could not induce thrombosis without vein ligation in rats, the enhancement of thrombogenesis by sulfatide in the in vivo model might require endothelial damage and/or venous congestion, both of which could be induced by vein ligation.