Sandra Schiffman

Preparation, Characterization, and Activation of a Highly Purified Factor XI: Evidence that a Hitherto Unrecognized Plasma Activity Participates in the Interaction of Factors XI and XII

Summary. Highly purified native factor XI has been prepared from normal plasma using a five step purification scheme. Purified factor XI is essentially free of factors II, V, VII, VIII, IX, X, XII, and Fletcher factor. No plasminogen‐plasmin could be detected. Purified factor XI decays rapidly but can be stabilized with human serum albumin. Factor XI migrates between the β and γ globulins on starch block electrophoresis. It has an apparent molecular weight on gel filtration of 210 000. Purified factor XI is activated by weak trypsin. No detectable BAEe esterase activity accompanies this activation. Neither factor XII adsorbed to kaolin nor activated factor XII in solution could activate purified factor XI; both reagents activate factor XI in dilute factor XII deficient plasma. Hence, plasma appears to supply a third activity which facilitates the interaction of factors XI and XII. This activity is shown to be distinct from Fletcher factor.

Partial purification and characterization of contact activation cofactor.

The contact phase of intrinsic clotting involves Factor XI, Factor XII, Fletcher factor, and a fourth activity that we call contact activation cofactor (CAC). All four of these activities are reduced or absent in Dicalite-adsorbed plasma. A modified activated partial thromboplastin time assay for CAC has been defined by using a substrate of Dicalite-adsorbed plasma combined with partially purified sources of Factors XI and XII, and Fletcher factor. The following properties of CAC in plasma have been determined by using the assay: it is stable up to 60 min at 56 degrees C; gradually loses activity at 80 degrees C; is stable between pH 6 and 9; is precipitated by ammonium sulfate between 40% and 50% saturation; is slightly adsorbed by A1(OH)3; and is eluted from DEAE-cellulose after the major protein peaks. A purification procedure has been devised that separates CAC from other known clotting factors. Isolated CAC was less stable than CAC in plasma, but in the presence of dilute human serum albumin it retained full activity for 80 min at 56 degrees C. On gel filtration CAC had an apparent mol wt of 220,000 daltons. These properties are consistent with those described for Fitzgerald factor, which further supports the conclusion that CAC and Fitzgerald factor represent the same activity. Isolated CAC promoted the generation of activated Factor XI (XIa) in a mixture containing purified Factor XI, Factor XII, and kaolin. The amount of Factor XIa generated was proportional to the amount of added CAC. No time-consuming reaction between Factor XI or Factor XII and CAC could be demonstrated.

Formation of Intrinsic Factor-X-Activator Activity, with Special Reference to the Role of Thrombin

Summary When thrombin‐activated purified human factor VIII (factor VIIIt), purified human factor IXa, lipid and calcium were combined, a very rapid reaction occurred involving the lipid and at least one of the plasma clotting factors. No other interaction between the components of intrinsic factor‐X activator could be demonstrated. Full activity developed so rapidly as to suggest an instantaneous reaction. In contrast, when native factor VIII was used instead of factor VIIIt, insignificant factor‐X activator was found over 15 min. Further experiments with hirudin confirmed that a preliminary activation of factor VIII is an absolute requirement for the development of human intrinsic factor‐X activator activity. Both thrombin and trypsin effectively activated factor VIII, which suggests that activation results from proteolysis. Factor IXa, factor XIa, factor Xa, collagen, connective tissue, platelet fractions, plasmin and Reptilase ‘R’could not activate factor VIII within a physiologically significant time interval.

Platelets and initiation of intrinsic clotting.

Summary. Comparison of activities in platelet rich and platelet poor plasmas from normal donors and patients deficient in either factor VIII, IX, XI or XII indicates that platelets contain activities which can partially substitute for plasma factors XI and XII. The factor‐XI‐like activity is expressed in a one‐stage activated partial thromboplastin assay and in an intact prothrombin consumption system. The factor‐XII‐like activity is scarcely detectable in a one‐stage assay but markedly enhances the defective prothrombin consumption of factor XII deficient plasma. Intact prothrombin consumption tests with platelet poor plasmas fortified with cephalin show that in the presence of high concentrations of platelet factor 3 activity only trace contact activation is required to promote good prothrombin consumption. The platelet, by supplying both platelet factor 3 and activities bypassing plasma contact activation factors XI and XII, may provide an important route for activating intrinsic clotting.

Separation of plasma thromboplastin antecedent (PTA) and Hageman factor (HF) from human plasma.

Summary 1) Hageman factor and plasma thromboplastin antecedent have been separated from human plasma and each other by ion exchange chromatography. 2) The defect in “exhausted plasma” is not corrected by purified PTA, purified HF, or a combination of the two.

The Mandatory Role of Lipid in the Interaction of Factors VIII and IX.

Summary 1. An interaction between activated Factor IX and Factor VIII which is independent of Factor X has been confirmed using human reagents. The product, activated Factor VIII, is a very labile activity. 2. Lipid has been shown to be a mandatory cofactor in the interaction of Factor VIII and serum factors in the presence of thrombin.

Factor XI and Platelets: Evidence that Platelets Contain only Minimal Factor XI Activity and Antigen

Factor‐XI activity of platelets has been studied in platelet‐rich plasmas and isolated platelet suspensions. Fresh platelets in both environments had little or no measurable factor‐XI activity. Frozen and thawed platelet‐rich normal plasma had markedly elevated apparent factor‐XI activity and factor‐IX activity as compared to platelet‐poor plasma. Frozen and thawed platelet‐rich and platelet‐poor normal plasmas had equivalent factor‐XI antigen. Platelets isolated from normal blood and from factor‐XI deficient blood had the same small amounts of apparent factor‐XI activity, which increased slightly on freezing and thawing. The data indicates that minimal factor XI is associated with the platelet. The markedly elevated apparent factor‐XI activity of frozen and thawed platelet‐rich plasma is shown to reflect the interaction of a platelet activator with plasma clotting factors to produce a later activated‐clotting‐intermediate.

Purification and characterization of platelet factor XI

Factor XI activity and antigen was purified about 300 fold from human platelets through chromatography on Con-A Sepharose, SP-Sephadex C-50, immobilized goat anti-factor XI, and SP-Sephadex. The partially purified platelet factor XI (Pt-XI) could be activated by activated factor XII generated in situ from single chain factor XI in a reaction requiring high molecular weight kininogen (HMWK) and a surface. Native Pt-XI migrated as a molecule of Mr = 245,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as identified by Western blotting. On reduction, Pt-XI appeared to have a Mr = 52,000. Neither form was affected by exposure to trypsin. Incubation of Pt-XI with purified factor XII, HMWK, and kaolin produced activated platelet factor XI clotting activity and, concomitantly, the generation over time of a new chain on reduced SDS-PAGE of Mr = 44,500. The coagulant activity of the activated form could be neutralized by diisopropyl flurophosphate (DFP). Incubation of the activated mixture with 3H-DFP followed by reduced SDS-PAGE showed the active site to be associated with a unit of Mr = 44,500. The adsorption domain as defined by adsorption to kaolin was localized to the Mr = 44,500 chain containing the active site. Hence, both active site and adsorption functions, properties of separate chains in plasma factor XI, reside in the same chain of Mr = 44,500 of platelet factor XI.

Phospholipids accelerate factor IX activation by surface bound factor XIa

Activation of bovine factor IX by surface bound factor XIa which was generated either by activation of human citrated factor IX deficient plasma or a mixture of purified human factors XII, high molecular weight kininogen (HMWK) and XI in glass tubes, is accelerated by cephalin. Human brain cephalin in dilutions ranging from 1:5 to 1:500 was studied for its effect on the activation of factor IX in concentrations of 1.0 u/ml and 16 u/ml. Cephalin dilutions from 1:5 to 1:30 accelerated the activation of the concentrated factor IX sample two- to threefold. Protein cleavage of this factor IX sample in the presence of 1:30 cephalin occurred twice as fast as in the absence of cephalin. Activation of the dilute factor IX sample (1.0 u/ml) was most effectively accelerated by cephalin in dilutions from 1:30 to 1:250. In all experiments the presence of phospholipid led to an increased factor IX cleavage concomitantly with faster generation of factor IXa activity. The results demonstrate that phospholipids actively participate in blood coagulation at an earlier stage than previously described.

Increased Factor VIII Levels in Suspected Carriers of Hemophilia A Taking Contraceptives by Mouth

THE probability that a potential carrier of hemophilia A is a true carrier can be determined from her factor VIII level.1 By chance, we measured factor VIII activity in 2 potential carriers when th...