Pharmacologic targeting of coagulation factors XII and XI by monoclonal antibodies reduces thrombosis in nitinol stents under flow.

BACKGROUND: Cardiovascular implantable devices, such as vascular stents, are critical for the treatment of cardiovascular diseases. However, their success is dependent on robust and often long-term antithrombotic therapies. Yet, the current standard-of-care therapies often pose significant bleeding risks to patients. Coagulation factor (F)XI and FXII have emerged as potentially safe and efficacious targets to safely reduce pathologic thrombin generation in medical devices. OBJECTIVES: To study the efficacy of monoclonal antibody-targeting FXII and FXI of the contact pathway in preventing vascular device-related thrombosis. METHODS: The effects of inhibition of FXII and FXI using function-blocking monoclonal antibodies were examined in a nonhuman primate model of nitinol stent-related thrombosis under arterial and venous flow conditions. RESULTS: We found that function-blocking antibodies of FXII and FXI reduced markers of stent-induced thrombosis in vitro and ex vivo. However, FXI inhibition resulted in more effective mitigation of thrombosis markers under varied flow conditions. CONCLUSION: This work provides further support for the translation of contact pathway of coagulation inhibitors for their adjunctive clinical use with cardiovascular devices.

Factor XI Deficiency.

Factor XI (FXI) deficiency (hemophilia C or Rosenthal disease) was first described in the 1950s in a multigenerational family experiencing bleeding related to surgery and dental procedures. Managing patients with FXI deficiency presents several challenges, including a lack of correlation of bleeding symptoms with FXI activity levels, the large volume of fresh frozen plasma required to achieve hemostatic FXI levels, lack of availability of FXI concentrate in certain regions of the world, and the inherent thrombotic risk associated with replacement therapy. This article summarizes presentation, diagnosis, and management of patients with FXI deficiency in a variety of clinical settings.

Characterization of hereditary factor XI deficiency in Taiwanese patients: identification of three novel and two common mutations.

Hereditary coagulation factor XI (FXI) deficiency is a rare bleeding disorder, but information on FXI deficiency in Taiwanese patients remains scarce. We evaluated clinical and genetic features of severe FXI deficiency patients in Taiwan. We collected clinical information and performed coagulation laboratory tests and genetic studies in ten unrelated Taiwanese families with severe FXI deficiency. FXI coagulation activity was assayed using a one-stage method. FXI antigen was determined using enzyme-linked immunosorbent assay. Underlying genetic mutations were evaluated using direct sequencing methods. Ten unrelated Taiwanese patients with hereditary FXI deficiency and variable bleeding tendencies were analyzed. Half of the patients were male. The most common bleeding manifestations were easy bruising (40%), bleeding after dental procedures (40%), and postoperative bleeding (33%). Two patients (20%) were asymptomatic. No correlation was found between bleeding manifestations and baseline FXI levels. Three novel mutations were identified: c.1322delT p.Lys442Cysfs*8, c.599G > C p.Cys200Ser, and IVS4 c.325 + 2del124. Two common mutations, c.1107C > T p.Tyr369* (40%) and c.841C > T p.Gln281* (30%), were also found. No correlation existed between bleeding and FXI activity, highlighting the difficulty in predicting FXI deficiency-related bleeding. Three novel FXI genetic mutations and two common mutations were identified, contributing to the known spectrum of FXI deficiency-related mutations.

Activation of rabbit Hageman factor by homogenates of cultured rabbit endothelial cells.

Rabbit Hageman factor was proteolytically cleaved and activated by a homogenate prepared from cultured rabbit endothelial cells. Cleavage of radiolabeled Hageman factor was monitored by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Endothelial cell-mediated cleavage of Hageman factor was demonstrated both in a purified system and in plasma, was time and concentration dependent, and was associated with formation of the characteristic 28,000 M(r) form of active Hageman factor. The rate of cleavage of Hageman factor was not affected by Triton X-100 (Rohm and Haas, Co., Philadelphia, Pa.), hexadimethrine bromide (Polybrene, Aldrich Chemical Co., Inc., Milwaukee, Wis.), hirudin, soybean trypsin inhibitor, or antisera to plasminogen or prekallikrein. However, cleavage was enhanced by kaolin, and was inhibited by diisopropyl-fluorophosphate. The enzyme responsible for cleavage of Hageman factor was localized to the 100,000-g-sedimentable, subcellular fraction of the endothelial cell homogenate and was relatively specific, because neither radiolabeled rabbit Factor XI nor rabbit prekallikrein were themselves proteolytically cleaved by the endothelial cell homogenate. However, when these molecules were incubated with the homogenate in the presence of Hageman factor, both Factor XI and prekallikrein were cleaved, demonstrating that Hageman factor had been activated by the endothelial cell homogenate. Furthermore, the kallikrein generated by endothelial cell homogenate-activated Hageman factor was capable of liberating kinin from high molecular weight kininogen as measured by bioassay. Cultured rabbit endothelial cells, therefore, possess the capacity to activate Hageman factor by proteolysis. This may be one mechanism for Hageman factor activation in vivo.

Plasma levels of bradykinin are suppressed in factor XII-deficient mice.

A genetically-transmissible factor (F) XII-inactivated allele has been produced in mice by targeted replacement of exons 3-8 of the FXII gene with the neomycin resistance gene. Interbreeding of these mice provided offspring homozygous for two inactivated FXII alleles (FXII(-/-)). Male and female FXII-deficient mice bred normally in all genotypic combinations of the heterozygous and homozygous states, and the offspring survived to adulthood, suggesting that a total FXII deficiency does not affect embryonic development and survival. Neither FXII transcripts nor FXII antigen was found in various tissues of adult FXII(-/-) mice. No obvious unchallenged coagulopathies were present in FXII(-/-) adult mice, despite greatly prolonged activated partial thromboplastin times in this mouse cohort. FXII(-/-) mice were then used to assess the in vivo importance of the plasma FXII/prekallikrein/kininogen pathway in provision of resting plasma bradykinin (BK) levels and in generation of plasma BK stimulated by contact with an artificial surface, using a new and greatly improved plasma BK assay developed during these studies. It was found that approximately 50% of resting BK, and all of the contact-stimulated plasma BK, was provided by this FXII-dependent pathway, without a requirement for FXI. These results provide clear evidence that surface-stimulated BK production, in mice, is dependent on the activation of FXII.

Hemophilia A and B are associated with abnormal spatial dynamics of clot growth.

To gain greater insight into the nature of the bleeding tendency in hemophilia, we compared the spatial dynamics of clotting in platelet-free plasma from healthy donors and from patients with severe hemophilia A or B (factor VIII:C or IX:C<1%). Clotting was initiated via the intrinsic or extrinsic pathway in a thin layer of nonstirred plasma by bringing it in contact with the glass or fibroblast monolayer surface. The results suggest that clot growth is a process consisting of two distinct phases, initiation and elongation. The clotting events on the activator surface and the preceding period free of visible signs of clotting are the initiation phase. In experiments with and without stirring alike, this phase is prolonged in hemophilic plasma activated by the intrinsic, but not the extrinsic pathway. Strikingly, both hemophilia A and B are associated with a significant deterioration in the elongation phase (clot thickening), irrespective of the activation pathway. The rate of clot growth in hemophilic plasma is significantly lower than normal and declines quickly. The resulting clots are thin, which may account for the bleeding disorder.

Effect of hemodialysis on contact group of coagulation factors, platelets, and leukocytes.

Earlier reports have suggested possible activation and consumption of factor XII during hemodialysis. To investigate this possibility, a series of in vivo and in vitro experiments were conducted using different dialysis membranes and two different dialysates (acetate and bicarbonate). Factors XII and XI activities, factor XII concentration, and high-molecular-weight kininogen were measured. In addition, platelet count, white blood cell count, and hematocrit were monitored. Contrary to the previous reports, no discernible consumption of factor XII, factor XI, or high-molecular-weight kininogen was found irrespective of the type of membrane or the composition of the dialysate used. Transient leukopenia was noted with cellulosic membranes, whereas none occurred with polyacrylonitrile dialyzers. The composition of dialysate did not affect the white blood cell count during dialysis.

Surface adsorption of factor XI. II. Evidence that different mechanisms are involved in binding to glass and plastic materials.

Blood coagulation factors XI and XIa possess binding site(s) for glass and plastics, located in the heavy chain of the molecule. To elucidate the nature of binding, adsorption and desorption properties of factor XI and XIa to different surfaces have been studied. Desorption experiments with high salt (2.4 M NaC1) suggest participation of ionic forces in the binding to glass. This is consistent with the decreased adsorption of factor XI (pI-9.0) to glass with increasing pH. The non-ionic detergent Triton X-100, which splits hydrophobic bonds, desorbs factor XI very well from plastics and partially from glass. The anionic detergent SDS, which will split hydrophobic as well as ionic bonds, is the most effective agent tested for the elution of factor XI from glass. We, therefore infer, that the binding of factor XI to glass is the combined effect of ionic and hydrophobic binding, whereas the adsorption of factor XI to plastics is primarily hydrophobic.

Surface independent factor XI activation by thrombin in the presence of high molecular weight kininogen.

A deficiency of one of the proteins of the contact system of blood coagulation does not result in a bleeding disorder. For this reason activation of blood coagulation via this system is believed to be an in vitro artefact. However, patients deficient in factor XI do suffer from variable bleeding abnormalities. Recently, an alternative pathway for factor XI activation has been described. Factor XI was found to be activated by thrombin in the presence of dextran sulfate as a surface. However, high molecular weight kininogen (HK), to which factor XI is bound in plasma, and fibrinogen were shown to block this activation suggesting it to be an in vitro phenomenon. We investigated the thrombin-mediated factor XI activation using an amplified detection system consisting of factors IX, VIII and X, which was shown to be very sensitive for factor XIa activity. This assay is approximately 4 to 5 orders of magnitude more sensitive than the normal factor XIa activity assay using a chromogenic substrate. With this assay we found that factor XI activation by thrombin could take place in the absence of dextran sulfate. The initial activation rate was approximately 0.3 pM/min (using 25 nM factor XI and 10 nM thrombin). The presence of dextran sulfate enhanced this rate about 8500-fold. A very rapid and complete factor X activation was observed in the presence of dextran sulfate. Although only minute amounts of factor XIa were formed in the absence of dextran sulfate, significant activation of factor X was detected in the amplification assay within a few minutes.(ABSTRACT TRUNCATED AT 250 WORDS)

Factor XI activation by meizothrombin: stimulation by phospholipid vesicles containing both phosphatidylserine and phosphatidylethanolamine.

The activation of factor XI by meizothrombin was investigated using recombinant meizothrombin (R155A meizothrombin) that is resistant to autocatalytic removal of fragment 1. Meizothrombin was capable of activating factor XI at an activation rate similar to that of thrombin. Dextran sulphate and heparin, known cofactors of thrombin-mediated factor XI activation, did not stimulate the activation of factor XI by meizothrombin. However, the activation of factor XI by meizothrombin was markedly enhanced by vesicles containing phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylethanolamine (PE), whereas PC/PS or PC/PE vesicles only had a minor effect on the activation. Thrombin-mediated factor XI activation was not influenced by phospholipids. The effect of PC/PS/PE and PC/PS vesicles was studied in a factor XI dependent clot lysis assay. In this assay, factor XI inhibits clot lysis by a feedback loop in the intrinsic pathway via thrombin-mediated factor XI activation. Removal of endogenous phospholipids in plasma by centrifugation resulted in an increased clot lysis, which could be restored to the pre-centrifugation level by the addition of PC/PS/PE vesicles, but not by PC/PS vesicles. When clot lysis was initiated by factor IXa in the presence of a factor XIa blocking antibody, there was no difference in inhibitory effect of PC/PS/PE or PC/PS vesicles. These data suggested that the differences in clot lysis inhibition observed between PC/PS/PE and PC/PS vesicles were caused by factor XI activation by meizothrombin. Meizothrombin-mediated factor XI activation may therefore play an important role in the antifibrinolytic feedback loop in the intrinsic pathway.

Coagulation on biomaterials in flowing blood: some theoretical considerations.

Are truly inert biomaterials feasible? Recent mathematical models of coagulation which are reviewed here suggest that such materials are impossible. This conclusion, which is certainly consistent with our collective experimental evidence, arises from the calculation that conversion of Factor XI to XIa never drops to zero even at the highest flow rates and with virtually no Factor XIIa bound to a surface. Residual amounts of XIa are still formed which can in principle kick-off the coagulation cascade. Furthermore, if the flow rates and corresponding mass transfer coefficients are low and in spite of these near-vanishing levels of the initiating coagulants, the surprising result is that substantial amounts of thrombin are produced. On the contrary, under slightly higher flow conditions, there can be more substantial levels of initiating coagulants, yet paradoxically thrombin production is near zero. This article presents a theoretical understanding of the events which take place during the interaction of biomaterials with flowing blood. We follow these events from the time of first contact to the final production of thrombin. The effect of flow and surface activity on the contact phase reactions is examined in detail and the two are found to be intertwined. The common pathway is also examined and here the main feature is the existence of three flow dependent regions which produce either high or very low levels of thrombin, as well as multiple thrombin steady states. In a final analysis we link the two segments of the cascade and consider the events which result. In addition, we note that multiple steady states arise only in the presence of two (thrombin) feedback loops. Single loops or the bare cascade will produce only single steady states. With some imagination one can attribute to the feedback loops the role of providing the cascade with a mechanism to produce high thrombin levels in case of acute need (e.g. bleeding) or to allow levels to subside to 'stand-by' when there is no need for clotting. We present this as a partial answer to the question: Why is the coagulation cascade so complex and what is the importance of the feedback loops?

Anticoagulant mechanism of sulfonated polyisoprenes.

The influence of sulfonated polyisoprene (SPIP) on coagulation factors and human blood cells was investigated to elucidate and compare its anticoagulant mechanism with that of heparin. While the number of red cells was unaffected, the number of platelets decreased dramatically in the presence of SPIP due to aggregation. Using a synthetic peptide substrate to assay thrombin activity in the presence of its natural inhibitor, antithrombin (AT), we observed no stimulation by SPIP of AT-mediated inhibition. Nevertheless, thrombin cleavage of its natural substrate fibrinogen to fibrin peptide A was slightly inhibited. SPIP altered the electrophoretic mobility of fibrinogen and completely inhibited fibrinogen from clotting. We detected no significant influence of SPIP on factors II, VII, IX, and X, while factor XI and factors V and VIII were only slightly affected. Therefore, the main mechanism of SPIP's anticoagulant activity appears to be a strong interaction with fibrinogen and fibrin monomer, first, to prevent proteolytic conversion of the former to the latter and second, to inhibit polymerization of the fibrin monomer, once formed.

Partial thromboplastin time in the presence of heparin: a rapid polybrene neutralization method.

The detection of heparin in plasma and its neutralization to permit assessment of the prolonged partial thromboplastin time is a constant need in the clinical laboratory. A rapid, simple method which utilizes Polybrene after the initial contact activation step is described and compared to the standard neutralization procedures. The present method has the advantage of not requiring Polybrene titration and of requiring only small volumes of plasma making it particularly valuable in pediatric patients.

Interactions of antithrombin and proteins in the plasma contact activation system with immobilized functional heparin.

The interactions of antithrombin (AT) and the contact phase clotting factors with two commercially available heparinized surfaces are reported. The Carmeda (CBAS) and Corline surfaces along with controls (a sulfonated polyethylene surface and a CBAS analog in which the heparin used was devoid of specific AT-binding sequences) were exposed to human plasma. Adsorbed proteins were eluted and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The CBAS and Corline surfaces adsorbed large amounts of AT, whereas adsorption on the controls was negligible. Immunoblots for the four contact phase clotting factors indicated less contact activation on the CBAS and Corline surfaces than on the controls. Determination of adsorbed functional AT using a FXa inhibition assay showed that the CBAS surface adsorbed about 4 times as much AT as the Corline surface. Adsorption of AT to the control surfaces was minimal. Assays for adsorbed FXII and FXIIa based on kallikrein generation showed that all four surfaces adsorbed similar amounts of FXII. However, on the controls, most of the FXII was in activated form, whereas on the CBAS and Corline surfaces very little activation occurred.

Abnormal plasma clot structure and stability distinguish bleeding risk in patients with severe factor XI deficiency.

BACKGROUND: Factor XI (FXI) deficiency is a rare autosomal recessive disorder. Many patients with even very low FXI levels (< 20 IU dL(-1) ) are asymptomatic or exhibit only mild bleeding, whereas others experience severe bleeding, usually following trauma. Neither FXI antigen nor activity predicts the risk of bleeding in FXI-deficient patients. OBJECTIVES: (i) Characterize the formation, structure and stability of plasma clots from patients with severe FXI deficiency and (ii) determine whether these assays can distinguish asymptomatic patients ('non-bleeders') from those with a history of bleeding ('bleeders'). METHODS: Platelet-poor plasmas were prepared from 16 severe FXI-deficient patients who were divided into bleeders or non-bleeders, based on bleeding associated with at least two tooth extractions without prophylaxis. Clot formation was triggered by recalcification and addition of tissue factor and phospholipids in the absence or presence of tissue plasminogen activator and/or thrombomodulin. Clot formation and fibrinolysis were measured by turbidity and fibrin network structure by laser scanning confocal microscopy. RESULTS: Non-bleeders and bleeders had similarly low FXI levels, normal prothrombin times, normal levels of fibrinogen, factor VIII, von Willebrand factor and factor XIII, and normal platelet number and function. Compared with non-bleeders, bleeders exhibited lower fibrin network density and lower clot stability in the presence of tissue plasminogen activator. In the presence of thrombomodulin, seven of eight bleeders failed to form a clot, whereas only three of eight non-bleeders did not clot. CONCLUSIONS: Plasma clot structure and stability assays distinguished non-bleeders from bleeders. These assays may reveal hemostatic mechanisms in FXI-deficient patients and have clinical utility for assessing the risk of bleeding.

Factor XI anion-binding sites are required for productive interactions with polyphosphate.

BACKGROUND: Conversion of factor XI (FXI) to FXIa is enhanced by polymers of inorganic phosphate (polyP). This process requires FXI to bind to polyP. Each FXIa subunit contains anion-binding sites (ABSs) on the apple 3 (A3) and catalytic domains that are required for normal heparin-mediated enhancement of FXIa inhibition by antithrombin. AIMS: To determine the importance of FXI ABSs to polyP enhancement of FXI activation. METHODS: Recombinant FXI variants lacking one or both ABSs were tested in polyP-dependent purified protein systems, plasma clotting assays, and a murine thrombosis model. RESULTS: In the presence of polyP, activation rates for FXI lacking either ABS were reduced compared with wild-type FXI, and FXI lacking both sites had an even greater defect. In contrast to heparin, polyP binding to FXIa did not enhance inhibition by antithrombin and did not interfere with FXIa activation of FIX. FXI lacking one or both ABSs does not reconstitute FXI-deficient plasma as well as wild-type FXI when polyP was used to initiate coagulation. In FXI-deficient mice, FXI lacking one or more ABSs was inferior to wild-type FXI in supporting arterial thrombus formation. CONCLUSIONS: The ABSs on FXIa that are required for expression of heparin's cofactor activity during protease inhibition by antithrombin are also required for expression of polyP cofactor activity during FXI activation. These sites may contribute to FXI-dependent thrombotic processes.

Immobilized transition metal ions stimulate contact activation and drive factor XII-mediated coagulation.

BACKGROUND: Upon contact with an appropriate surface, factor XII (FXII) undergoes autoactivation or cleavage by kallikrein. Zn(2+) is known to facilitate binding of FXII and the cofactor, high molecular weight kininogen (HK), to anionic surfaces. OBJECTIVES: To investigate whether transition metal ions immobilized on liposome surfaces can initiate coagulation via the contact pathway. METHODS AND RESULTS: Liposomes containing a metal ion-chelating lipid, 1,2-dioleoyl-sn-glycero-3-{(N[5-amino-1-carboxypentyl]iminodiacetic acid)succinyl} ammonium salt (DOGS-NTA), were prepared by membrane extrusion (20% DOGS-NTA, 40% phosphatidylcholine, 10% phosphatidylserine, and 30% phosphatidylethanolamine). Ni(2+) immobilized on such liposomes accelerated clotting in normal plasma, but not factor XI (FXI)-deficient or FXII-deficient plasma. The results were similar to those obtained with a commercial activated partial thromboplastin time reagent. Charging such liposomes with other transition metal ions revealed differences in their procoagulant capacity, with Ni(2+) > Cu(2+) > Co(2+) and Zn(2+). Plasma could be depleted of FXI, FXII and HK by adsorption with Ni(2+) -containing beads, resulting in longer clot times. Consistent with this, FXI, FXII and HK bound to immobilized Ni(2+) or Cu(2+) with high affinity as determined by surface plasmon resonance. In the presence of Ni(2+) -bearing liposomes, K(m) and k(cat) values derived for autoactivation of FXII and prekallikrein, as well as for activation of FXII by kallikrein or prekallikrein by FXIIa, were similar to literature values obtained in the presence of dextran sulfate. CONCLUSIONS: Immobilized Ni(2+) and Cu(2+) bind FXII, FXI and HK with high affinity and stimulate activation of the contact pathway, driving FXII-mediated coagulation. Activation of the contact system by immobilized transition metal ions may have implications during pathogenic infection or in individuals exposed to high levels of pollution.