P A McKee

Substructure of human von Willebrand factor.

Using electron microscopy, we have visualized the substructure of human von Willebrand factor (vWf) purified by two different approaches. vWf multimers, which appear as flexible strands varying in length up to 2 micron, consist of dimeric units (protomers) polymerized linearly in an end-to-end fashion through disulfide bonds. Examination of small multimers (e.g., one-mers, two-mers, and three-mers) suggests that each protomer consists of two large globular end domains (22 X 6.5 nm) connected to a small central node (6.4 X 3.4 nm) by two flexible rod domains each approximately 34 nm long and approximately 2 nm in diameter. The protomer is 120 nm in length when fully extended. These same structural features are seen both in vWf molecules that were rapidly purified from fresh plasma by a new two-step procedure and in those purified from lyophilized intermediate-purity Factor VIII/vWf concentrates. The 240,000-mol wt subunit observed by gel electrophoresis upon complete reduction of vWf apparently contains both a rod domain and a globular domain and corresponds to one half of the protomer. Two subunits are disulfide-linked, probably near their carboxyl termini, to form the protomer; disulfide bonds in the amino-terminal globular ends link promoters to form vWf multimers. The vWf multimer strands have at least two morphologically distinct types of ends, which may result from proteolytic cleavage in the globular domains after formation of large linear polymers. In addition to releasing fragments that were similar in size and shape to the repeating protomeric unit, plasmic degradation of either preparation of vWf reduced the size of multimers, but had no detectable effect on the substructure of internal protomers.

The effect of fibrin-stabilizing factor on the subunit structure of human fibrin

The formation of human fibrin from fibrinogen has been examined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, a method which separates a mixture of proteins on the basis of differences in molecular weight. It has been found that the plasma from a patient with a congenital deficiency of fibrin-stabilizing factor forms clots lacking the cross links among the alpha- and gammachains found in normal, cross-linked human fibrin. The addition of purified fibrin-stabilizing factor or normal plasma to the deficient plasma results in extensive cross-linking of the chains. Thus, the fibrinogen in the fibrin-stabilizing factor deficient plasma appears to be normal and forms fibrin which contains dimeric, cross-linked gamma-chains and polymeric, high molecular weight forms of alpha-chains. By the use of these electrophoretic methods, it has also been possible to develop a highly sensitive method for measuring the content of fibrin-stabilizing factor in plasma. This method depends upon the use of urea-treated fibrinogen, which is completely devoid of fibrin-stabilizing factor, but which forms the usual cross-linked subunits after conversion to fibrin by thrombin in the presence of fibrin-stabilizing factor.

Demonstration and characterization of specific binding sites for factor VIII/von Willebrand factor on human platelets.

The presence of specific Factor VIII/von Willebrand factor (FVIII/vWF) binding sites on human platelets has been demonstrated by using 125I-FVIII/vWF and washed human platelets. Binding is ristocetin-dependent and increases in proportion to the concentration of ristocetin from 0.2 to 1 mg/ml. Binding of 125I-FVIII/vWF to platelets can be competitively inhibited by unlabeled human or bovine FVIII/vWF, but not by human thrombin, fibrinogen, alpha 2-macroglobulin, equine collagen, or a lectin of Ricinus communis. Scatchard analysis of binding data indicated that the dissociation constant of FVIII/vWF receptors is 0.45--0.5 nM. There are 31,000 binding sites per platelet at 1 mg/ml of ristocetin concentration. The optimal pH range for binding is from 7.0 to 7.5. At a concentration of 2 mM, EGTA inhibits 86% of the binding; however, 20 mM of Ca++, Mg++, or EDTA have little effect. Binding sites for FVIII/vWF were found only on platelets, and no significant binding was detected with human erythrocytes or polymorphonuclear leukocytes.

The subunit polypeptides of human fibrinogen

Abstract Three polypeptide chains have been isolated from S -sulfofibrinogen by chromatography on carboxymethylcellulose. The three chains, designated A, B, and C, were pure as judged by ultracentrifugal analysis and zone electrophoresis. Each chain corresponded to one of the three electrophoretic components observed in unfractionated S -sulfofibrinogen. By the methods of sedimentation-equilibrium analysis in the ultracentrifuge, the molecular weights of the chains were estimated to be 47,000 for the A-chain, 56,000 for the B-chain, and 63,500 for the C-chain. The amino acid composition and tryptic peptide pattern of each chain was unique. If it is assumed that human fibrinogen contains one pair of each of the unique chains, then the molecular weight and amino acid composition of fibrinogen are closely accounted for by the weights and composition of the chains.

The Subunit Structure of Normal and Hemophilic Factor VIII

Human factor VIII from normals and hemophiliacs was partially purified by ethanol and polyethylene glycol precipitations. Final purification was achieved by gel filtration on 2 or 4% agarose or ion exchange chromatography on diethylaminoethyl cellulose. Comparable amounts of highly purified protein were obtained from normal and hemophilic plasma following the agarose chromatography step. Highly purified factor VIII was not dissociated by 6 M guanidine hydrochloride or 1% sodium dodecyl sulfate. However, when reduced by beta-mercaptoethanol and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, a single subunit species with an estimated 195,000 molecular weight was found for both normal and hemophilic factor VIII. By sedimentation equilibrium analysis, the normal factor VIII subunit was homogeneous and had an estimated molecular weight of 202,000. The subunit polypeptides from normal or hemophilic factor VIII contained carbohydrate. Each was homogeneous by isoelectric focusing. Immunodiffusion of purified normal and hemophilic factor VIII against rabbit antiserum to purified normal human factor VIII showed a single line of precipitation. Very low concentrations of purified human thrombin initially increased the activity of normal factor VIII about threefold and then progressively destroyed activity by 3 h. Only minimal activation occurred with hemophilic factor VIII. Both the activation and inactivation of normal and hemophilic factor VIII were unaccompanied by detectable changes in subunit molecular weight. These findings may have implications for the definition of the molecular defect in hemophilic factor VIII.

Mechanism of Ancrod Anticoagulation A DIRECT PROTEOLYTIC EFFECT ON FIBRIN

Fibrin formed in response to ancrod, reptilase, or thrombin was reduced by beta-mercaptoethanol and examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It was found that ancrod progressively and totally digested the alpha-chains of fibrin monomers at sites different than plasmin; however, further digestion of fibrin monomers by either reptilase or thrombin was not observed. Highly purified ancrod did not activate fibrin-stabilizing factor (FSF); however, the reptilase preparation used in these experiments, like thrombin, activated FSF and thereby promoted cross-link formation. Fibrin, formed by clotting purified human fibrinogen with ancrod, reptilase, or thrombin for increasing periods of time in the presence of plasminogen, was incubated with urokinase and observed for complete lysis. Fibrin formed by ancrod was strikingly more vulnerable to plasmin digestion than was fibrin formed by reptilase or thrombin. The lysis times for fibrin formed for 2 hr by ancrod, reptilase, or thrombin were 18, 89, and 120 min, respectively. Evidence was also obtained that neither ancrod nor reptilase activated human plasminogen. These results indicate that fibrin formed by ancrod is not cross-linked and has significantly degraded alpha-chains: as expected, ancrod-formed fibrin is markedly susceptible to digestion by plasmin.

Electron microsocpy of plasmic fragments of human fibrinogen as related to trinodular structure of the intact molecule.

We have examined rotary shadowed, purified plasmic fragments of human fibrinogen with the electron microscope and have determined the relation of these fragments to the intact fibrinogen molecule. Both intact fibrinogen and its earliest cleavage product, fragment X, are trinodular. The next largest product, fragment Y, consists of two linked nodules. The two terminal products, fragments D and E, are single nodules. From measurements of simultaneously shadowed specimens of these different species, we conclude that the outer nodules of the trinodular fibrinogen molecule are the fragment D-containing regions and the central nodule is the fragment E-containing region.

Some Effects of Calcium on the Activation of Human Factor VIII/Von Willebrand Factor Protein by Thrombin

When Factor VIII/von Willebrand factor (FVIII/vWF) protein is rechromatographed on 4% agarose in 0.25 M CaCl(2), the protein and vWF activity appear in the void volume, but most of the FVIII procoagulant activity elutes later. Recent evidence suggests that the delayed FVIII procoagulant activity is a proteolytically modified form of FVIII/vWF protein that filters anomalously from agarose in 0.25 M CaCl(2). To test whether or not thrombin is the protease involved, the effect of 0.25 M CaCl(2) on FVIII/vWF and its reaction with thrombin was examined. About 30% of the FVIII procoagulant activity was lost immediately when solutions of FVIII/vWF protein were made 0.25 M in CaCl(2). When FVIII in 0.15 M NaCl was activated with 0.04 U thrombin/ml and then made 0.25 M in CaCl(2), the procoagulant activity of a broad range of FVIII/vWF protein concentrations remained activated for at least 6 h. But, in 0.25 M CaCl(2), the increase in FVIII procoagulant activity in response to thrombin was much more gradual and once activated, the procoagulant activity was stabilized by 0.25 M CaCl(2). When thrombin-activated FVIII/vWF protein was filtered on 4% agarose in 0.15 M NaCl, there was considerable inactivation of FVIII procoagulant activity; however, the procoagulant activity that did remain eluted in the void volume. In contrast, when thrombin-activated FVIII/vWF protein was filtered in 0.25 M CaCl(2), the FVIII procoagulant activity eluted well after the void volume and remained activated for 6 h. The procoagulant peak isolated by filtering nonthrombin-activated FVIII/vWF protein on agarose in 0.25 M CaCl(2) was compared to that isolated from thrombin-activated FVIII/vWF protein. Both procoagulant activity peak proteins had about the same specific vWF activity as the corresponding void volume protein. Before reduction, the sodium dodecyl sulfate gel patterns for the two procoagulant activity peak proteins were the same. After reduction, the gel pattern for the nonthrombin-activated procoagulant activity peak protein contained bands of 195,000, 148,000-120,000, 79,000, 61,000, 51,000, and 18,000 daltons whereas the pattern for the reduced thrombin-activated procoagulant activity peak protein always lacked the higher molecular weight bands, but consistently showed the four lower molecular weight bands to be well resolved. Taken together, these results imply that thrombin generates the FVIII procoagulant activity that is stabilized by 0.25 M CaCl(2) and elutes aberrantly from 4% agarose in that solvent.

Comparison of ancrod and heparin as anticoagulants following endarterectomy in the dog.

An experimental model of surgically-induced arterial thrombosis was devised using the femoral arteries of dogs. Within 7 days, 67% of the arteries became completely thrombosed and only 12% remained completely patent. In the group of dogs that received low-dose heparin, 69% of the vessels were completely thrombosed and 6% remained completely patent. In the group of dogs treated with low-dose Ancrod to induce partial defibrination, 75% remained completely patent while only 19% of their femoral arteries were completely thrombosed. Although the ancrod was effective in preventing arterial thrombosis, 88% of the wounds showed moderate to severe separations. Most likely the absence of a fibrin lattice, necessary for the securement and growth of fibroblasts as the wound heals, explains this latter effect. Thus while Ancrod may become useful as an anticoagulant in certain clinical situations, it should not be used in proximity to surgery. Finally, in these studies of acute arterial thromboses, low-dose heparin therapy offered no protective effect.

FIBRIN FORMATION AND DISSOLUTION

Abstract: Proteolysis is not only important in the generation of thrombin, but also in the last phase of blood coagulation, i.e., fibrin formation. Fibrin dissolution and removal is also accomplished by proteolysis. Thrombin cleaves amino terminal peptides from fibrinogen as well as from the zymogen form of fibrin-stabilizing factor in setting the stage for fibrin polymerization. The activation of the plasma protein, plasminogen, by urokinase occurs by the cleavage of two peptide bonds to form the tryptic-like enzyme, plasmin. Both fibrinogen and fibrin are progressively digested by plasmin to give degradation products, some of which have functional importance as inhibitors of fibrin polymerization. Characterization studies of the fibrinogen/fibrin degradation products have provided clues to the structures of fibrinogen and fibrin.