Bonno N. Bouma

Misfolded proteins activate Factor XII in humans, leading to kallikrein formation without initiating coagulation

When blood is exposed to negatively charged surface materials such as glass, an enzymatic cascade known as the contact system becomes activated. This cascade is initiated by autoactivation of Factor XII and leads to both coagulation (via Factor XI) and an inflammatory response (via the kallikrein-kinin system). However, while Factor XII is important for coagulation in vitro, it is not important for physiological hemostasis, so the physiological role of the contact system remains elusive. Using patient blood samples and isolated proteins, we identified a novel class of Factor XII activators. Factor XII was activated by misfolded protein aggregates that formed by denaturation or by surface adsorption, which specifically led to the activation of the kallikrein-kinin system without inducing coagulation. Consistent with this, we found that Factor XII, but not Factor XI, was activated and kallikrein was formed in blood from patients with systemic amyloidosis, a disease marked by the accumulation and deposition of misfolded plasma proteins. These results show that the kallikrein-kinin system can be activated by Factor XII, in a process separate from the coagulation cascade, and point to a protective role for Factor XII following activation by misfolded protein aggregates.

Inactivation of kallikrein in human plasma.

Human plasma kallikrein is inactivated by plasma protease inhibitors. This study was designed to determine the nature of these protease inhibitors and to assess their relative importance in the inactivation of kallikrein. Therefore, the kinetics of kallikrein inactivation and the formation of kallikrein inhibitor complexes were studied in normal plasma and in plasma depleted of either alpha 2-macroglobulin (alpha 2M), C1 inhibitor, or antithrombin (AT III). Prekallikrein was activated by incubation of plasma with dextran sulfate at 4 degrees C. After maximal activation, kallikrein was inactivated at 37 degrees C. Inhibition of kallikrein amidolytic activity in AT III-deficient plasma closely paralleled the inactivation rate of kallikrein in normal plasma. The inactivation rate of kallikrein in alpha 2M-deficient plasma was slightly decreased compared with normal plasma, but in contrast to normal, C1 inhibitor-deficient, and AT III-deficient plasma, no kallikrein amidolytic activity remained after inactivation that was resistant to inhibition by soybean trypsin inhibitor. Suppression of kallikrein activity in C1 inhibitor-deficient plasma was markedly decreased, and this was even more pronounced in plasma deficient in both C1 inhibitor and alpha 2M. The pseudo first-order rate constants for kallikrein inactivation in normal, AT III-deficient, alpha 2M-deficient, C1 inhibitor-deficient plasma, and plasma deficient in both alpha 2M and C1 inhibitor, were 0.68, 0.60, 0.43, 0.07, and 0.016 min-1, respectively. Sodium dodecyl sulfate gradient polyacrylamide slab gel electrophoresis showed that during inactivation of kallikrein in plasma, high-Mr complexes were formed with Mr at 400,000-1,000,000, 185,000, and 125,000-135,000, which were identified as complexes of 125I-kallikrein with alpha 2M, C1 inhibitor, and AT III, respectively. In addition, the presence of an unidentified kallikrein-inhibitor complex was observed in AT III-deficient plasma. 52% of the 125I-kallikrein was associated with C1-inhibitor, 35% with alpha 2M, and 13% with AT III and another protease inhibitor. A similar distribution of 125I-kallikrein was observed when the 125I-kallikrein inhibitor complexes were removed from plasma by immunoadsorption with insolubilized anti-C1 inhibitor, anti-alpha 2M, or anti-AT III antibodies. These results suggest that only covalent complexes are formed between kallikrein and its inhibitors in plasma. As a function of time, 125I-kallikrein formed complexes with C1 inhibitor at a higher rate than with alpha 2M. No difference was observed between the inactivation rate of kallikrein in high-Mr kininogen-deficient plasma and that in high-Mr kininogen-deficient plasma reconstituted with high-Mr kininogen; this suggests that high-Mr kininogen does not protect kallikrein from inactivation in the plasma milieu. These results have quantitatively demonstrated the major roles of C1 inhibitor and alpha 2M in the inactivation of kallikrein in plasma.

Thrombin activatable fibrinolysis inhibitor (TAFI) at the interface between coagulation and fibrinolysis.

The thrombin-catalysed conversion of plasma fibrinogen into fibrin and the development of an insoluble fibrin clot are the final steps of the coagulation cascade during haemostasis. A delicate balance between coagulation and fibrinolysis determines the stability of the fibrin clot. Thrombin Activatable Fibrinolysis Inhibitor (TAFI) plays an important role in this process. TAFI is activated by thrombin and protects the fibrin clot against lysis. The role of TAFI in bleeding and thrombotic disorders is discussed as well as its novel emerging role in wound healing and inflammation.

Immunocytochemical localization of fibrinogen, platelet factor 4, and beta thromboglobulin in thin frozen sections of human blood platelets.

Affinity-purified monospecific antibodies against human fibrinogen and the platelet-specific proteins platelet factor 4 and beta thromboglobulin were used to localize these antigens in thin and ultra-thin frozen sections of mildly fixed, washed human blood platelets. By immunofluorescent double-labeling experiments the distribution of fibrinogen was compared to that of platelet factor 4 and beta thromboglobulin. All three antigens occurred in virtually all platelets and showed and identical, dotlike distribution. For immunoelectron microscopy we used protein A-colloidal gold on ultra-thin frozen sections to visualize the specific reaction indirectly. The staining for platelet factor 4, beta thromboglobulin, and fibrinogen localized exclusively over alpha-granules of washed platelets. Within the granules, platelet factor 4 was localized preferentially in the electron dense, alpha-granule nucleoid, whereas fibrinogen was more predominant in the electron-lucent granule periphery. Beta thromboglobulin localization did not show a preferential intragranular distribution.

Identification of fibronectin type I domains as amyloid-binding modules on tissue-type plasminogen activator and three homologs

The serine protease tissue-type plasminogen activator (tPA), a key enzyme in hemostasis, is activated by protein aggregates with amyloid-like properties. tPA is implicated in various pathologies, including amyloidoses. A major task is to further elucidate the mechanisms of amyloid pathology. We here show that the fibronectin type I domain of tPA mediates the interaction with amyloid protein aggregates. We found that in contrast to full-length tPA, a deletion-mutant of tPA, lacking the first three N-terminal domains (including the fibronectin type I domain), fails to activate in response to amyloid protein aggregates. Using recombinantly produced domains of tPA in direct binding assays, we subsequently mapped the amyloid-binding region to the fibronectin type I domain. This domain co-localized with congophylic plaques in brain sections from patients with Alzheimers disease. Fibronectin type I domains from homologous proteases factor XII, hepatocyte growth factor activator and from the extracellular matrix protein fibronectin also bound to aggregated amyloidogenic peptides. Finally, we demonstrated that the isolated fibronectin type I domain inhibits amyloid-induced aggregation of blood platelets. The identification of the fibronectin type I domain as an amyloid-binding module provides new insights into the (patho-) physiological role of tPA and the homologous proteins which may offer new targets for intervention in amyloid pathology.

Plasma protein S contains zinc essential for efficient activated protein C-independent anticoagulant activity and binding to factor Xa, but not for efficient binding to tissue factor pathway inhibitor

Protein S (PS) is a cofactor for activated protein C (APC), which inactivates coagulation factors (F) Va and Villa. Deficiency of protein C or PS is associated with risk of thrombosis. We found that PS also has APC‐independent anticoagulant activity (PS‐direct) and directly inhibits thrombin generated by FXa/FVa (prothrombinase complex). Here we report that PS contains Zn2+ that is required for PS‐direct and that is lost during certain purification procedures. immunoaffinity‐purified PS contained 1.4 ± 0.6 Zn2+/mol, whereas MonoQ‐purified and commercial PS contained 0.15 ± 0.15 Zn2+/mol. This may explain the controversy regarding the validity of PS‐direct. Zn2+ content correlated positively with PS‐direct in prothrombinase assays and clotting assays, but APC‐cofactor activity of PS was indepen‐dent of Zn2+ content. PS‐direct and Zn2+ were restored to inactive PS under mildly denaturing conditions. Conversely, o‐phenanthroline reversibly impaired the PS‐direct of active PS. Zn2+‐containing PS bound FXa more efficiently (Xdapp = 9.3 nM) than Zn2+‐deficient PS (Xdapp=110 nM). PS bound TFPI efficiently, independently of Zn2+ content (Xdapp = 21 nM). Antibodies that block PS‐direct preferentially recognized Zn2+‐containing PS, suggesting conformation differences at or near the interface of 2 laminin G‐like domains near the PS C terminus. Thus, Zn2+ is required for PS‐direct and efficient FXa binding and may play a role in stabilizing PS conformation.—Heeb, M. J., Prashun, D., Griffin, J. H., Bouma, B. N. Plasma protein S contains zinc essential for efficient activated protein C‐independent anticoagulant activity and binding to factor Xa, but not for efficient binding to tissue factor pathway inhibitor. FASEB J. 23, 2244–2253 (2009)

Decreased plasma sensitivity to activated protein C by oral contraceptives is associated with decreases in plasma glucosylceramide

Summary.u2002 Oral contraceptive (OC) use increases venous thrombosis (VTE) risk and causes activated protein C (APC) resistance. Plasma glucosylceramide (GlcCer) deficiency is associated with VTE and GlcCer functions as an APC anticoagulant cofactor. Because estradiol decreases GlcCer in cultured cells, we hypothesized OC use would decrease plasma GlcCer and contribute to APC resistance. In a pilot study, seven female adults alternatively took second and third generation OCs and plasma samples were analyzed for GlcCer using high performance liquid chromatography and for APC sensitivity using modified prothrombin time assays. Second and third generation OC usage decreased the APC sensitivity ratio by 8.1%u2003±u20034.7% (Pu2003=u20030.004) and 11.7%u2003±u20038.2% (Pu2003=u20030.013) and plasma GlcCer levels by 10.1%u2003±u20036.8% (Pu2003=u20030.008) and 11.0%u2003±u20035.1% (Pu2003=u20030.002), respectively. The plasma GlcCer level correlated with the sensitivity of plasma to APC (Pu2003=0.017, ru2003=u20030.51, nu2003=u200321 plasma samples). Thus, both second and third generation OC usage decreased plasma GlcCer which could cause a reduction in the plasma sensitivity to APC/protein S, thereby potentially increasing VTE risk.

Characterization of a variant prekallikrein, prekallikrein Long Beach, from a family with mixed cross-reacting material-positive and cross-reacting material-negative prekallikrein deficiency.

Studies of plasma prekallikrein in a family with prekallikrein deficiency were made. Three children had no clotting activity but approximately 35% antigen levels, and the mother and five children had twice as much prekallikrein antigen as clotting activity, suggesting the presence of a dysfunctional molecule. A nonfunctional variant form of prekallikrein was purified that contained no prekallikrein clotting activity. The variant and normal molecules were both 80,000 mol wt, immunologically indistinguishable and complexed similarly with high molecular weight kininogen. Isoelectric focusing studies suggested a difference of one charged amino acid residue. The variant was cleaved by beta-Factor XIIa 200 times slower than the normal molecule, and no amidolytic activity was detected for the cleaved variant. These data and other observations suggest that an amino acid was substituted in the variant near the NH2-terminal end of the kallikrein light chain resulting in slower cleavage by beta-Factor XIIa and the absence of enzymatic activity.

Unraveling the mystery of von Willebrand factor

*Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA; Laboratory of Thrombosis andHaemostasis, Department of Haematology, University Medical Centre Utrecht; and Department of Blood Coagulationand Department of Plasma Proteins, Sanquin, Amsterdam, the NetherlandsTo cite this article: Bouma BN, van Mourik JA. Unraveling the mystery of von Willebrand factor. J Thromb Haemost 2006; 4: 489–95.