Robert H. Wagner

Factor XIII in human plasma and platelets

Plasma and platelet factor XIII levels were measured in normal human donors and in a patient congenitally deficient in factor XIII. The purpose of these experiments was to study the role of platelet factor XIII in blood coagulation. On polyacrylamide disc electrophoresis, factor XIII activity in extracts of washed normal platelets appeared as a single peak. This peak was missing or very low when factor XIII-deficient platelet extract was used. The patient was also studied before and after transfusion of fresh frozen plasma. Before transfusion, factor XIII activity could not be detected in the patients plasma or platelet extracts. 24 hr after transfusion the plasma factor XIII level was still at the anticipated value, but factor XIII activity could not be detected in the platelet extracts. These experiments imply that plasma factor XIII was not transported across the platelet membrane in measurable quantities in vivo. This suggests that platelet factor XIII is a true platelet component and originates in the platelet precursor, the megakaryocyte. Thrombelastographic studies suggest that platelet factor XIII participates in the coagulation process. Thrombelastograms of factor XIII-deficient samples had decreased maximum amplitude and clot elasticity values. The abnormalities were fully corrected by the addition of washed normal platelets to factor XIII-deficient plasma; preincubation of the normal platelets in the deficient plasma had no additional effect. This indicates that platelet factor XIII is immediately available during clot formation.

DEMONSTRATION OF FACTOR XIII IN HUMAN MEGAKARYOCYTES BY A FLUORESCENT ANTIBODY TECHNIQUE

This work presents direct evidence for the cytoplasmic location of clotting Factor XI11 in human bone marrow megakaryocytes. This indicates that blood platelets derive Factor XI11 through de novo synthesis rather than through plasmatic transport across the platelet membranes. Evidence from plasma Factor XI11 assays of thrombocytopenic patients indicates that a major source of plasma Factor XI11 can be outside the platelet or megakaryocyte. The possible role of platelet Factor XI11 in the hemostatic process is discussed.

Effects of thrombin treatment of preparations of factor VIII and the Ca2+-dissociated small active fragment.

When human, canine, or bovine factor VIII preparations are chromatographed on 4% agarose at ionic strength 0.2, the factor VIII activity elutes as a single peak in the void volume with slight tailing. Incubation of such preparations with dilute (0.01 U/ml) highly purified thrombin results in some activation of factor VIII. Chromatography of such incubation mixtures, under the same conditions as before, results in elution of two peaks of factor VIII activity one in the void volume and one much later with marked tailing. The void volume peak has most of the protein and some factor VIII activity. These void volume fractions also contain all the von Willebrand factor activity of thrombin-treated bovine preparations. Longer treatment with thrombin, or treatment with stronger thrombin, appears to shift much more of the procoagulant activity to the later eluting peak. Also, when the peak of factor VIII activity, found in the void volume after thrombin treatment, was again incubated with dilute thrombin, an increase in factor VIII activity occurred. Chromatography of this incubation mixture demonstrated only a small amount of activity in the void volume, while the bulk of the activity was present in the second peak. On the other hand, thrombin treatment of factor VIII activity from peak 2 caused a rapid decline of activity instead of a further increase. It is proposed that the residual factor VIII activity found in the void volume represents unreacted factor VIII, while the late eluting peak represents thrombin-activated material that is of smaller apparent size. The late eluting peak differs from the small active factor VIII fragment obtained by Ca2+ dissociation, as the latter can be activated by thrombin. A similar set of experiments was performed using ultracentifugation of bovine factor VIII preparations on sucrose density gradients. Results of these experiments agreed completely with those obtained with get chromatography. Preparations made from human hemophilic plasma, by the procudure employed in the purification of human factor VIII, were also incubated with thrombin and chromatographed. von Willebrand factor was again found only in the void volume fractions, but there was no factor VIII activity in any fractions eluted. In other control experiments, activated and unactivated factor VIII fractions did not clot fibrinogen and contained no assayable factor IX or X. The thrombin-modified factor VIII of small size was inactivated by both a naturally occurring human inhibitor to factor VIII and the gamma globulin fraction of a rabbit antisera produced against the calcium-dissociated small active factor VIII fragment.

Sedimentation of plasma antihemophilic factor

Abstract Purified bovine plasma antihemophilic factor (AHF) preparations and canine plasma were sedimented in a swinging bucket type rotor of a preparative ultracentrifuge. A simple technique is described for sampling contents of the tubes for analysis after sedimentation. Sedimentation diagrams and S 20,w values are presented for AHF and fibrinogen at 4 and 21 °C. It is shown that AHF in canine plasma probably exists in an associated form, either as a polymer or associated with fibinogen. Apparently different degrees of association exist in bovine fractions, with the degree of association depending upon the ionic strength of the solution. The S 20,w value of 4.3 S probably represents the sedimentation coefficient for the AHF monomer.

A Naturally Occurring Antibody That Inhibits Fibrin Polymerization

A 13-year-old girl with chronic aggressive hepatitis, postnecrotic cirrhosis, ulcerative colitis, and a coagulation defect acquired an antibody that specifically interfered with fibrin formation. We sought to characterize the antibody and determine the mechanism of its inhibitory activity. The patients purified fibrinogen was functionally normal; however, the antibody inhibited the self-assembly of fibrin and prolonged the clotting times of the patients plasma. This antibody, which belonged to the IgG class of immunoglobulins, acted early in the polymerization process to inhibit the association of fibrin monomers, as indicated by a prolonged lag time and a decreased slope in the polymerization curves. It did not inhibit fibrinopeptide cleavage or fibrin cross-linking. Affinity chromatography indicated that the antibody bound strongly to both fibrinogen and fibrin monomer.

The Defect in Hemophilic and von Willebrand's Disease Plasmas Studied by a Recombination Technique

Factor VIII in preparations from normal plasma is a large glycoprotein of greater than 2 million molecular weight which elutes in the exclusion volume of 4% agarose gels at an ionic strength of 0.15. Recent studies have demonstrated that the factor VIII in canine and bovine plasma is a macromolecular complex composed of a large inert carrier protein and a noncovalently bound small fragment which contains the procoagulant active site. This complex is dissociable in 0.25 M CaCl(2), and conditions for its recombination have been reported. The present study reports the dissociation characteristics of normal human factor VIII preparations in 0.25 M CaCl(2) and the ability to achieve quantitative recombination of the dissociated fragments of normal human and bovine factor VIII after the removal of Ca(2+). The recombination technique was used to characterize further the defect in hemophilia and von Willebrands disease. Void volume preparations from human hemophilia A(-), canine hemophilia A, and human von Willebrands plasma, with no factor VIII procoagulant activity, were mixed with the small active fragment prepared from the normal plasma of their respective species. Chromatography of the three mixtures in agarose gel showed that the fractions from the human hemophilic plasmas contained a molecule that bound the small active normal fragment, but neither the fractions from canine hemophilia A plasmas nor the fractions from the human von Willebrands plasmas demonstrated evidence of such material. These data suggest that there is present in human hemophilia A plasma a normal functional carrier molecule which is absent or nonfunctional in the plasma of hemophilic dogs and humans with von Willebrands disease.

EFFECT OF SULFHYDRYL INHIBITORS ON PLATELET AGGLUTINABILITY.

Summary 1. In the presence of Ca++ or Mg++, ADP induces agglutination of platelets washed at 4°C. 2. The sulfhydryl inhibitors PCMB, NEM, and MMN inhibit the ability of washed canine platelets to agglutinate with thrombin or ADP in the presence of Ca++. 3. The effectiveness of PCMB in inhibiting platelet agglutinability is dependent upon thrombin concentration used to induce agglutination. The inhibitory action of PCMB is independent of changes in ADP concentration. 4. MMN causes deagglutination of platelets from clumps formed in the presence of 1.0 U/ml thrombin and Ca++, if the MMN is added to the platelet clumps within 30 seconds of agglutination. 5. MMN, at non-inhibitory concentrations (2.0 × 10-5 m or less), induces rapid and intense platelet agglutination in the presence of Ca++.

Antihemophilic Factor (AHF) Levels Following Transfusions of Blood, Plasma and Plasma Fractions.∗

Summary 1. Canine and human hemophilic subjects were transfused with homologous blood or plasma, Dogs were also transfused with AHF-rich plasma fractions. The increase in plasma AHF levels following transfusions was proportional to the amount of AHF administered. Over half of the AHF activity disappeared within a few hours, although traces of AHF may persist for nearly a week. Frequent replacement transfusions are required if AHF levels as high as 5-10% are to be maintained. 2. Injection of potent plasma fractions into normal dogs resulted in transient supernormal AHF levels. Half of the injected AHF was lost in about 2 hours. 3. The minimum amount of AHF required to give normal values is not uniform with different clotting tests. AHF levels above 1% resulted in normal values for the clotting time and prothrombin utilization tests. Levels above 15% resulted in normal values for the PTT test.

Platelet agglutination and deagglutination with a sulfhydryl inhibitor, methyl mercuric nitrate: Relationships to platelet ATPase

Abstract The effects of methyl mercuric nitrate (MMN) on platelet ATPase activity and on agglutination and deagglutination of washed canine platelets were studied. Through use of “aging” platelets, the platelet agglutinating activity of MMN was compared with that of ADP, thrombin, and trypsin. Methyl mercuric nitrate, in a concentration that produced platelet agglutination, enhanced platelet ATPase activity in the presence of calcium. A concentration of MMN, which previously was demonstrated to inhibit ADP and thrombin-induced platelet agglutination, either failed to enhance or was inhibitory to ATPase activity. By the use of 2 × 10 −4 M MMN, platelet agglutination induced by thrombin and Ca ++ was separated into a stage characterized by ready deagglutination and a stage with incomplete or no observable deagglutination. Washed canine platelets, “aged” at 28°C, retained their agglutinability with MMN as well as with thrombin and trypsin after they would no longer agglutinate with ADP. The presence of resinated, BaSO 4 -adsorbed plasma in the final agglutination systems prolonged the agglutinability of “aging” washed platelets with both ADP and MMN, but failed to equalize the two systems. It is suggested that the agglutinating and deagglutinating properties of weak and strong MMN are related to platelet ATPase activity and changes in platelet repellent force.

Preparation of bovine platelet aggregating factor (PAF).

Abstract A simple four step procedure for the preparation of bovine platelet aggregating factor (PAF)1 is described. The final product is obtained in good yield. After reduction, the product gives one main band with polyacrylamide sodium dodecyl sulfate (SDS) disc electrophoresis that stains for protein and carbohydrate and has an approximate molecular weight of 260,000. Before reduction, electrophoresis of the product on two percent agarose in SDS shows a series of high molecular weight aggregates ranging in molecular weight from 1.15 to 12 million.