Robert F. Baugh

Effect of exercise on the factor VIII complex: a correlation of the Von Willebrand antigen and factor VIII coagulant antigen increase.

Abstract Ten volunteers ran 3/4 miles as fast as possible and blood was drawn prior to and immediately following the exercise. Plasma factor VIII coagulant activity rose an average of 187% while the factor VIII coagulant antigen rose only an average of 107% and correlated better with the two-stage clotting assay (102%) and the von Willebrand factor antigen (115%). It is concluded that the disproportionate rise in factor VIII procoagulant activity above the von Willebrand factor is observed only when using the one-stage factor VIII clotting assay.

Evidence that factor VIII and the ristocetin aggregating factor (VIIIRist) are separate molecular entities.

The most highly purified preparations of human factor VIII have at least three clearly defined properties. They shorten the clotting time of hemophilic plasma, an activity which will for convenience be referred to as VIIIcoag; they give a precipitin reaction with a rabbit antibody prepared against the preparation, a property referred to as the factor VIII related-antigen (VIIIR-Ag); and in the presence of the antibiotic, Ristocetin, they correct the defective aggregation of platelets found in von Willebrands disease (1-4), an activity which will be referred to as VIIIRist. There are two prevailing concepts: (a) that each property represents a function of the same molecule, (b) that there is more than one molecule, each with one or more properties (5). The purpose of the experiments described below was to determine whether VIIIcoag, VIIIRist, and VIIIR-Ag reside on the same or on different molecules. The addition of a naturally occurring human factor VIII antibody known to be of the IgG class neutralizes VIIIcoag without affecting the expression of VIIIRist or VIIIR-Ag (3). Rabbit antibodies, on the other hand, prepared against highly purified factor VIII preparations neutralize VIIIRist and VIIIR-Ag (3), but their VIIIcoag neutralizing activity may be relatively low (6, 7). These findings are not inconsistent with the one-molecule hypothesis as the antibody could neutralize one side chain carrying an active site without affecting others. If the one-molecule hypothesis is correct, following the neutralization of VIIIcoag or VIIIRist by the appropriate antibody, the addition of a second precipitating antibody directed against the IgG class should precipitate both VIIIcoag and VIIIRist activities bound to the first antibody; as the factor VIII molecule would carry all the active sites, the supernatant will be devoid of all the remaining properties of the molecule.

Separation of human factor VIII activity from the von willebrand's antigen and ristocetin platelet aggregating activity

Abstract Factor VIII coagulant activity was separated by ion-exchange chromatography from the von Willebrands antigen (Factor VIII-like antigen). The von Willebrands antigen was also separated from additional contaminating proteins and no longer aggregated washed human platelets in the presence of the antibiotic, ristocetin. Recombination of the von Willebrands antigen with these proteins restored restocetin-induced platelet aggregation. Highly purified human fibrinogen also restored ristocetin-dependent platelet aggregating activity to the purified von Willebrands antigen. The separated Factor VIII coagulant activity was unstable and lost >98% of the coagulant activity within three days at 0–4 °C. These findings show that Factor VIII preparations which demonstrate ristocetin-induced aggregation of washed human platelets contain at least three separable components.

The Passovoy Defect: Further Characterization of a Hereditary Hemorrhagic Diathesis

We studied a coagulation abnormality present in 12 members of five kindreds who bruised easily and bled excessively after minor trauma. Their activated partial thromboplastin times were between 32 and 39 seconds (normal, 22.8 to 28.8 seconds). Prothrombin times, thrombin times, platelet-function tests and the levels of factors XII, XI, IX, VIII, prekallikrein and high-molecular-weight kininogen were normal. Within these kindreds inheritance of prolonged partial thromboplastin times followed an autosomal and probably dominant pattern. The prolonged thromboplastin times were corrected by normal plasma and by normal plasma adsorbed with celite, but there was no mutual correction between plasmas of the patients. These subjects shared a common defect in the intrinsic pathway of coagulation that we designate by the probands surname, Passovoy.

Separation of Bovine Factor VIII-Related Antigen (Platelet Aggregating Factor) from Bovine Antihemophilic Factor

Summary It was shown that in bovine plasma, factor VHI-related antigen can be separated from factor VIII coagulant activity by chromatography on QAE Sephadex. The suggestion is made that the factor in bovine plasma which aggregates human platelets and which aggregates washed bovine platelets in the presence of ristocetin is identical to the factor VIII-like antigen.

Selective destruction of the platelet aggregation component in bovine factor VIII preparations by reduction

Abstract Treatment of a purified bovine factor VIII preparation with dithiothritol (5mM) irreversibly destroys the human platelet aggregation factor (PAF). The factor VIII level as measured by the one stage assay appears to be slightly activated. Sucrose density gradient ultracentrifugation yields factor VIII after reduction distinct from immunologically measured PAF. This suggests the reduced subunit size of factor VIII is smaller than PAF.

Substrate inhibition of the intrinsic generation of activated factor X (Stuart factor)

Abstract The generation of activated factor X by the product of the interaction of factors VIII and IXa in the presence of calcium and phosphatidyl serine was studied using the synthetic chromogenic substrate S-2222 (benzoylIleGluGlyArgp-nitroanilideHCl). For maximum generation of factor Xa, a ratio of greater than 100 units of factor VIII to 1 unit of factor IXa was required. Using purified bovine factor X, the Km for both the intrinsic and extrinsic conversion of factor X to Xa was found to be 0.20 μM - approximately 1.3 units/ml. The rate of factor Xa formation was inhibited at factor X levels greater than 3 units/ml. In contrast, the extrinsic generation of factor Xa using purified factor VII and tissue thromboplastin was not inhibited by factor X levels as high as 32 units/ml.

Effect of vancomycin on ristocetin and bovine PAF-induced agglutination of human platelets.

Abstract Vancomycin, an antibiotic similar in bacterial properties and chemical structure to ristocetin, unlike ristocetin does not induce von Willebrand factor-dependent agglutination of human platelets. Conversely, it inhibits this reaction in a competitive manner suggesting that both antibiotics compete for the same binding site which is responsible for the induction of agglutination. Vancomycin does not inhibit bovine platelet aggregating factor-induced platelet agglutination of human platelets. Thus ristocetin induced and bovine platelet aggregating factor induced agglutination can be distinguished on the basis of vancomycin inhibition. The mode of this inhibition can be explained by either of two different mechanisms. 1) If the site of action of ristocetin is the platelet membrane, then two distinct binding sites exist, one for the von Willebrand factor and one for bovine platelet aggregating factor. Present evidence does not favor this hypothesis. 2) If the initial site of action for ristocetin is the von Willebrand factor, then vancomycin competes with ristocetin for a binding site on the von Willebrand factor. The initial interaction of the von Willebrand factor. The initial interaction of the von Willebrand factor with ristocetin can then be divided into at least two distinct phases, an initial binding phase in which both vancomycin and ristocetin can participate, and a transformation phase which occurs with only ristocetin. The transformation phase results in the modification of the von Willebrand factor such that it binds to a pre-existing site on the platelet membrane.

The effect of pepsin solubilization on platelet aggregation by types I and III collagens.

The effect of pepsin solubilization on the platelet aggregating activity of type I collagen and type III collagen was examined. Pepsin-digested type I collagen was unable to initiate platelet aggregation in either soluble form or as pre-formed fibrils. In contrast, pepsin-digested type III collagen was active in soluble form or as preformed fibrils. Mixtures of type I and type III collagen were assayed for platelet aggregating activity. In soluble form, these mixtures demonstrated elevated activity with increasing type III concentration. When the mixtures were tested as pre-formed fibrils, the rate of aggregation was relatively constant with the combination 75% type III and 25% type I manifesting the highest activity. The lag time for onset of aggregation was also minimized for this same type III/type I ratio. The combinations of the two collagen types formed fibrils which reflected the amounts of types I and III collagens in solution.

Identification of platelet receptors for bovine von Willebrand factor on human platelets by a new platelet receptor test.

A new test for the quantitation of platelet receptor activity for bovine von Willebrand factor in human platelet extracts is described. The test is based on the competition for the von Willebrand factor occurring between platelets, an antibody against von Willebrand factor, and platelet extracts containing the von Willebrand receptor. Bovine von Willebrand factor aggregates human platelets directly. In the presence of EDTA, anti-bovine von Willebrand factor antibody disaggregates platelet aggregates induced by bovine von Willebrand factor. Disaggregation is measured by following the decrease in light transmission accompanying disaggregation. Platelet extracts inhibited the disaggregating activity of the antibody, and the percent inhibition was directly proportional to the amount of the platelet extract added. The decrease in platelet disaggregating activity of the antibody was used as an indicator of platelet receptor activity in a system consisting of platelet-rich plasma, bovine von Willebrand factor, antibody and platelet extract. With this method we found von Willebrand factor receptors in the whole platelet extract, the Triton X-100 platelet membrane extract and in platelet extract material which bound to a bovine Willebrand factor-Sepharose 6MB affinity column.