Role of human factor VIII in factor X activation.

The cofactor function of human Factor VIII in Factor X activation was investigated by an initial-rate assay of 3H-Factor X activation in the presence of human factor IXa, Ca2+, and either phospholipid or fresh washed human platelets. Purified Factor VIII that has not been activated by thrombin or Factor Xa supports Factor X activation after a lag of several minutes. A specific inhibitor of Factor Xa, which had no inhibitory activity against Factor IXa, markedly prolonged this lag, whereas specific thrombin inhibitors did not prolong the lag. These data support the conclusion that unactivated Factor VIII has no ability to support Factor X activation in a purified system until it is activated by Factor Xa feedback during the lag period. When Factor VIII was optimally preactivated by thrombin, the lag was completely abolished, regardless of the order of addition of the other reactants or the phospholipid source. These data indicate that there is no slow, time-dependent ordering of the reactants at the phospholipid or activated platelet surface if Factor VIII has been preactivated. Unactivated platelets did not support Factor X activation by Factors IXa and VIII. The effect of activated Factor VIII on the kinetics of bovine Factor X activation was primarily to increase the Vmax (54-fold), whereas with human Factor X, Factor VIII both increased the Vmax 56-fold and decreased the Km sixfold to 0.14 microM, similar to the plasma concentration of Factor X. Therefore, a change in the plasma factor X concentration would be expected to have a major effect on the rate of Factor X activation in vivo.     

Isolation and study of an acquired inhibitor of human coagulation factor V.

A coagulation Factor V inhibitor developed in a man 75 yr of age in association with an anaplastic malignancy and drug treatment (including the aminoglycoside antibiotic, gentamicin). The patient did not bleed abnormally, despite both surgical challenge and plasma Factor V activity of less than 1%. The inhibited plasma had grossly prolonged prothrombin and activated partial thromboplastin times, but a normal thrombin time. Mixing studies indicated progressive coagulation inhibition with normal plasma, but not with Factor V-deficient plasma, and reversal of coagulation inhibition by the addition of bovine Factor V to the patient's plasma. 1 ml of patient plasma inhibited the Factor V activity of 90 ml of normal human plasma. The inhibitor was isolated by sequential affinity chromatography on protein A-Sepharose and Factor V-Sepharose. The IgG isolate markedly inhibits the activity of prothrombinase assembled from purified Factors Xa and Va, calcium ion, and phospholipid vesicles, and partially inhibits prothrombinase assembled from purified Factor Xa, calcium ion, and normal platelets. The Factor V of platelets, however, appears relatively inaccessible to the antibody, inasmuch as platelets isolated from whole blood supplemented for 8 h with the antibody functioned normally with respect to platelet Factor V-mediated prothrombinase function. The absence of obvious hemorrhagic difficulties in the patient, the total inhibition of plasma Factor V by the inhibitor, and the apparent inaccessibility of platelet Factor V to the inhibitor specifically implicate platelet Factor V in the maintenance of hemostasis.     

Inhibition of the anticoagulant activity of protein S by prothrombin.

Protein S is a vitamin K-dependent protein cofactor to the anticoagulant, activated protein C (APC). This study examines the inhibition of human protein S anticoagulant activity by prothrombin. In the absence of protein S, the anticoagulant activity of APC measured in a Factor Xa recalcification time, was comparable using normal or plasma adsorbed with Al(OH)3. Protein S was an effective cofactor to APC in Al(OH)3-adsorbed plasma, but was significantly less active in normal plasma. Analysis of the difference in the two plasmas revealed that normal plasma contained an inhibitor to the anticoagulant activity of protein S that was removed by Al(OH)3 adsorption. Purification of this inhibitory activity demonstrated that it was mediated by the vitamin K-dependent protein, prothrombin. Prothrombin purified by conventional techniques caused immediate, dose-dependent inhibition of the cofactor activity of protein S in the presence of phospholipids or platelets, but had no effect on the anticoagulant activity of APC. The inhibition was demonstrable using a Factor Xa recalcification time, and studies of the rates of inactivation of purified Factor Va. Increasing concentrations of protein S overcame the inhibition by prothrombin and kinetic analysis of the interaction demonstrated that prothrombin acted as a competitive inhibitor to protein S. Immunoabsorption of prothrombin from plasma using immobilized antiprothrombin antibodies was associated with the complete removal of the protein S inhibitory activity. We conclude that the anticoagulant activity of protein S is modulated by prothrombin and that this may represent another regulatory mechanism of the natural anticoagulant system.     

Factor V (Quebec): a bleeding diathesis associated with a qualitative platelet Factor V deficiency.

Studies were performed on a French-Canadian family afflicted with a bleeding disorder exhibiting an autosomal dominant inheritance pattern and a severe bleeding diathesis after trauma. Clinical laboratory coagulation tests were unimpressive; the only persistent abnormalities include mild thrombocytopenia and moderately reduced Factor V clotting activities. Some individuals had prolonged Stypven times when platelet-rich plasma was used, suggesting that their platelets could not support functional prothrombinase complex assembly. Detailed studies were performed by use of plasma and isolated, washed platelets from a sister and brother. Bioassay data indicate that both individuals had Factor V activities of approximately 40 and 36% of normal, respectively. A comparison of the Factor V radioimmunoassay and bioassay data on the brother's plasma indicated that the circulating amount of Factor V functional activity was low relative to Factor V antigen concentration (approximately 65-75%). In both individuals, the platelet Factor V functional activities were extremely low (2-4%) relative to antigen levels present as determined by radioimmunoassay. These discrepancies between Factor V activities and antigen concentration do not appear to be due to an unstable Factor V molecule or to the presence of a Factor V or Factor Va inhibitor or inactivator. Kinetics of prothrombin activation by use of purified clotting factors indicated that thrombin-activated platelets from both individuals supported prothrombinase complex assembly identical to controls in the presence of added purified Factor Va. Consequently, their bleeding diathesis appears to reflect their platelet, rather than their plasma, Factor V activity. These results suggest that platelet Factor V is an essential component in maintaining stable and prolonged hemostasis after trauma.     

High-density lipoprotein enhancement of anticoagulant activities of plasma protein S and activated protein C

Low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol levels are associated, respectively, with either increased risk or apparent protective effects for atherothrombosis. The ability of purified LDL and HDL to downregulate thrombin formation, a contributor to atherothrombotic processes, was assessed. Purified HDL, but not LDL, significantly enhanced inactivation of coagulation factor Va by activated protein C (APC) and protein S, and HDL stimulated protein S–dependent proteolytic inactivation of Va by APC, apparently due to cleavage at Arg306 in Va. In normal plasma, added HDL enhanced APC/protein S anticoagulant activity in modified prothrombin-time clotting assays. When the anticoagulant potency of HDL was compared with phospholipid (PL) vesicles of well-defined composition using this assay, HDL appeared qualitatively different from PL vesicles because HDL showed only good anticoagulant activity, whereas PL vesicles were rather procoagulant. When 20 normal plasmas were tested using this clotting assay, apoA-I levels correlated with anticoagulant response to APC/protein S (r = 0.47, P = 0.035), but not with activated partial thromboplastin time–based APC resistance ratios. Because HDL enhances the anticoagulant protein C pathway in vitro, we speculate that HDL may help downregulate thrombin generation in vivo and that this anticoagulant action is one of HDL's beneficial activities.     

Detection and partial characterization of an inhibitor of plasminogen activator in human platelets

In this study, we demonstrate the presence of a previously undescribed fibrinolytic inhibitor in human serum. It has an apparent molecular weight of 50,000 and is not detected in serum derived from platelet-poor plasma, suggesting that it originates from platelets. This conclusion is supported by a number of observations. For example, extracts of washed, gel-filtered human platelets contain an inhibitor of similar activity and size, and physiological concentrations of thrombin induce its release from the platelets. Moreover, the kinetics and dose dependency of this release are similar to those observed for the release of platelet factor 4, and the release of both molecules is blocked by pretreating the platelets with prostaglandin E1 and theophylline. Mixing experiments, which were devised to investigate the specificity of the inhibitor, showed that the fibrinolytic activity initiated by both urokinase and tissue-type plasminogen activator was blocked by platelet releasate in a dose-dependent manner. In both cases, the amount of inhibition increased when the releasates were preincubated with the purified activators, indicating a direct interaction between the activators and an inhibitor(s). The inhibitory activity was removed by preincubating the releasates with antiserum prepared against an antiactivator purified from cultured bovine aortic endothelial cells. These results indicate that platelets contain an inhibitor which is released by thrombin, inhibits both urokinase and tissue-type plasminogen activator, and is immunologically similar to an inhibitor produced by endothelial cells. This molecule may represent a new class of inhibitors, the antiactivators, which function together with alpha 2-antiplasmin to regulate the fibrinolytic system of the blood. Its release from platelets by thrombin may protect the growing thrombus against premature dissolution initiated by plasminogen activators released by the endothelium.     

Proteolytic cleavage of protein S during the hemostatic response

BACKGROUND: Protein S is a vitamin K-dependent protein with anticoagulant properties. It contains a so-called thrombin-sensitive region (TSR), which is susceptible to cleavage by coagulation factor Xa (FXa) and thrombin. Upon cleavage, the anticoagulant activity of protein S is abolished. OBJECTIVE: The aim of the present study was to determine whether protein S is cleaved within the TSR during activation of the coagulation system under near physiological conditions. RESULTS: In a reconstituted coagulation system containing apart from protein S only procoagulant constituents and synthetic phospholipid vesicles, protein S was cleaved at Arg60 by the FXa generated (3 mol min(-1) mol(-1) enzyme). FXa-catalyzed cleavage of protein S, however, was inhibited by factor Va and prothrombin by more than 70%. During clotting of recalcified citrated plasma in the presence of a synthetic lipid membrane, no FXa-catalyzed proteolysis of protein S was observed. Substituting platelets for phospholipid vesicles resulted both in the reconstituted system and in plasma in cleavage of the TSR. Cleavage was at Arg60 and was observed upon platelet activation, irrespective of the presence of FXa (13 pmol min(-1) 10(-8) platelets). No cleavage by thrombin was observed in either the reconstituted coagulation system or clotting plasma. CONCLUSION: These findings suggest that in vivo the anticoagulant activity of protein S is not down-regulated by FXa or thrombin during activation of coagulation. Our results rather suggest a role for a platelet protease in down-regulating the anticoagulant activity of protein S during the hemostatic response.     

Thrombin-Induced Increase in Intracellular Cyclic 3′,5′-Adenosine Monophosphate in Human Platelets

The present data disagree with earlier suggestions that thrombin's effect on platelets is to cause a decrease in intracellular cyclic 3',5'-adenosine monophosphate. Washed human platelets or platelet-rich plasma were incubated at 37 degrees C with human thrombin. After centrifugation, the supernates were assayed for nucleotides and calcium released. The platelet pellets, and in some experiments the supernates as well, were assayed by radioimmunoassay for intracellular cyclic AMP. In the washed platelet system, increasing doses of thrombin to 0.5 U/cc induced increasing release of nucleotides and calcium. This was accompanied by an average twofold increase in intracellular cyclic AMP levels. Prostaglandin E(1), which inhibited 30-50% of release, induced a four- to fivefold increase in cyclic AMP levels that was additive to the cyclic AMP-stimulatory effect of thrombin. Theophylline, which inhibited only 20-40% of nucleotide release, was synergistic with thrombin in the intracellular accumulation of cyclic AMP. The time-course of cyclic AMP accumulation in response to thrombin was slower than thrombin-induced nucleotide release. Similar findings were made in the platelet-rich plasma system where thrombin stimulation of nucleotide release also resulted in a marked accumulation of intracellular cyclic AMP. Thrombin did not appear to stimulate the release of intracellular cyclic AMP.The mechanism underlying these observations was not apparent. The thrombin had no measurable inhibitory effect on platelet phosphodiesterase activity in either intact washed cells or the platelet homogenate supernates. Furthermore, thrombin inhibited, rather than stimulated, platelet adenyl cyclase activity in both intact washed cells and washed platelet particulate fractions. Of note, however, was the finding that thrombin did not completely inhibit the adenyl cyclase activity of prostaglandin-stimulated cells. Further work is needed to clarify the significance of this observation.Nonetheless, the accumulation of intracellular cyclic AMP in response to thrombin observed in the present study suggests that the antagonistic actions of various agents on the platelet release reaction, thought to underlie platelet function, may depend upon a mechanism more intricate than a straightforward mediation through directly opposite effects on platelet cyclic AMP.     

Human Platelets and Factor XI: LOCALIZATION IN PLATELET MEMBRANES OF FACTOR XI-LIKE ACTIVITY AND ITS FUNCTIONAL DISTINCTION FROM PLASMA FACTOR XI

Because human platelets participate in the contact phase of intrinsic coagulation and contain a Factor XI-like coagulant activity, the nature of the Factor XI-like activity was examined and compared with purified plasma Factor XI. The platelet factor XI-like activity was sedimented with the particulate fraction of a platelet lysate, was inactivated by heat (t(1/2) 3.5 min, 56 degrees C), was not a nonspecific phospholipid activity, and was destroyed by treatment with Triton X-100. Isolated platelet membranes were four-fold enriched in Factor XI activity and similarly enriched in plasma membrane marker enzymes. The Factor XI-like activity of platelet membranes was detected only when assayed in the presence of kaolin, which suggests that it is present in an unactivated form and can participate in contact activation. Concanavalin A inhibited the Factor XI-like activity of platelet lysates and platelet membranes but not of plasma or purified Factor XI. A platelet membrane-Factor XI complex was isolated after incubation of membranes with purified Factor XI. The Factor XI activity of the platelet membrane-plasma Factor XI complex was inhibited by concanavalin A, whereas unbound plasma Factor XI retained activity. An antibody raised against plasma Factor XI inhibited the in vitro Factor XI activity of plasma and of the platelet membrane-plasma Factor XI complex but had no effect on the endogenous Factor XI-like activity of washed lysed platelets or isolated platelet membranes. Washed platelets and isolated platelet membranes obtained from a Factor XI-deficient donor without a history of excessive bleeding had normal quantities of platelet Factor XI-like activity and normal behavior in the contact phase of coagulation (collagen-induced coagulant activity). These results indicate that platelet membranes contain an endogenous Factor XI-like activity that is functionally distinct from plasma Factor XI.     

Anticoagulant synergism of heparin and activated protein C in vitro. Role of a novel anticoagulant mechanism of heparin, enhancement of inactivation of factor V by activated protein C.

Interactions between standard heparin and the physiological anticoagulant plasma protein, activated protein C (APC) were studied. The ability of heparin to prolong the activated partial thromboplastin time and the factor Xa- one-stage clotting time of normal plasma was markedly enhanced by addition of purified APC to the assays. Experiments using purified clotting factors showed that heparin enhanced by fourfold the phospholipid-dependent inactivation of factor V by APC. In contrast to factor V, there was no effect of heparin on inactivation of thrombin-activated factor Va by APC. Based on SDS-PAGE analysis, heparin enhanced the rate of proteolysis of factor V but not factor Va by APC. Coagulation assays using immunodepleted plasmas showed that the enhancement of heparin action by APC was independent of antithrombin III, heparin cofactor II, and protein S. Experiments using purified proteins showed that heparin did not inhibit factor V activation by thrombin. In summary, heparin and APC showed significant anticoagulant synergy in plasma due to three mechanisms that simultaneously decreased thrombin generation by the prothrombinase complex. These mechanisms include: first, heparin enhancement of antithrombin III-dependent inhibition of factor V activation by thrombin; second, the inactivation of membrane-bound FVa by APC; and third, the proteolytic inactivation of membrane-bound factor V by APC, which is enhanced by heparin.     

Protein C supports platelet binding and activation under flow: role of glycoprotein Ib and apolipoprotein E receptor 2

Summary.  Background:  Activated protein C (APC) regulates thrombin generation and inhibits apoptosis. Endothelial protein C receptor (EPCR)-bound protein C is activated by thrombomodulin-bound thrombin. APC inactivates coagulation factors (F)Va/VIIIa and generates cytoprotective signaling downstream of protease-activated receptor-1 (PAR-1). Binding of APC to EPCR both modifies and induces PAR-1 signaling, but it is unknown if protein C interacts with cells in an alternative manner. Aim:  To determine whether platelets possess receptors for protein C that can generate intracellular signals. Results:  Immobilized protein C or APC supported platelet adhesion, lamellipodia formation and elevation of intracellular Ca²⁺. Adhesion of platelets to protein C or APC was inhibited by soluble recombinant apolipoprotein E receptor 2' (ApoER2') and by receptor-associated protein (RAP), an inhibitor of the low-density lipoprotein receptor family. Under shear, surface-bound protein C supported platelet adhesion and aggregation in a glycoprotein (GP)Ibα-dependent manner, and adhesion of platelets to immobilized protein C was abrogated by the addition of soluble forms of ApoER2' or RAP. APC bound to purified recombinant ApoER2' or GPIbα. Conclusions: Our data demonstrate that activation of platelets with rapid intracellular signaling caused by binding to immobilized protein C or APC occurs via mechanisms that require ApoER2 and GPIbα and that APC directly binds to purified ectodomains of the receptors ApoER2 and GPIbα. These findings imply that protein C and APC may directly promote cell signaling in other cells by binding to ApoER2 and/or GPIbα.     

Biosynthesis of factor V in isolated guinea pig megakaryocytes.

Although platelets contain Factor V, localized primarily in the alpha-granules, the origin of this coagulation cofactor in these cells is not known. We therefore explored whether isolated megakaryocytes could biosynthesize Factor V. Guinea pig plasma Factor V coagulant activity was demonstrated to be neutralized by human monoclonal and rabbit polyclonal antibodies directed monospecifically against human Factor V. These antibodies had been used earlier to purify human Factor V. These antibodies had been used earlier to purify human Factor V and to quantify Factor V antigen concentration, respectively (1983. Chiu, H. C., E. Whitaker, and R. W. Colman. J. Clin. Invest. 72:493-503). As determined by a competitive enzyme-linked immunosorbent assay with guinea pig plasma as a standard, Factor V solubilized from guinea pig megakaryocytes was present at 0.098 +/- 0.018 micrograms/10(5) cells. Each megakaryocyte contained about 500 times as much Factor V as is in a platelet (0.234 +/- 0.180 micrograms/10(8) platelets). The content of Factor V antigen in guinea pig plasma was greater (27.0 +/- 3.0 micrograms/ml) than that of Factor V antigen in human plasma (11.1 +/- 0.4 micrograms/ml). In contrast, human platelets contain ninefold more Factor V antigen (2.01 +/- 1.09 micrograms/10(8) platelets) than do guinea pig were 2.85 +/- 0.30 U/ml plasma, 0.022 +/- 0.012 U/10(8) platelets, and 0.032 +/- 0.03 U/10(5) megakaryocytes, compared with human values of 0.98 +/- 0.02 U/ml plasma and 0.124 +/- 0.064 U/10(8) platelets. Isolated megakaryocytes were found to contain Factor V by cytoimmunofluorescence. The megakaryocytes were incubated with [35S]methionine, and radiolabeled intracellular proteins purified were on a human anti-Factor V immunoaffinity column. The purified protein exhibited Factor V coagulant activity and neutralized the inhibitory activity of a rabbit antihuman Factor V antibody, which suggests that megakaryocyte Factor V is functionally and antigenically intact. These results indicate that Factor V is synthesized by guinea pig megakaryocytes. Nonetheless, megakaryocyte Factor V was more slowly activated by thrombin and in the absence of calcium was more stable after activation than was plasma Factor Va. Electrophoresis in sodium dodecyl sulfate and autoradiography of the purified molecule showed a major band of Mr 380,000 and a minor band of Mr 350,000, as compared with guinea pig and human plasma Factor V, where the protein had an Mr of 350,000. Both forms of Factor V were substrates for thrombin. Possible explanations for the higher molecular weight and different thrombin sensitivity and stability observed are that a precursor of Factor V was isolated or that the megakaryocyte Factor V had not been fully processed before isolation.     

Stimulation of Endothelial Cell Prostacyclin Production by Thrombin, Trypsin, and the Ionophore A 23187

Prostacyclin (PGI(2)) is an unstable prostaglandin which inhibits platelet aggregation and serotonin release and causes vasodilation. The PGI(2) activity produced by monolayers of cultured human endothelial cells and fibroblasts was measured by the ability of their supernates to inhibit platelet aggregation in platelet-rich plasma, or to inhibit thrombin-induced [(14)C]serotonin release from aspirin-treated, washed platelet suspensions. Monolayers of cultured human endothelial cells, stimulated with sodium arachidonate, thrombin, the ionophore A 23187, or trypsin, secreted PGI(2) into the supernatant medium. Monolayers of fibroblasts produced PGI(2) activity only when stimulated by arachidonate. "Resting," intact monolayers did not produce detectable PGI(2), nor did monolayers treated with ADP or epinephrine. Production of PGI(2) activity was abolished by treatment of the monolayers with indomethacin, tranylcypromine, or 15-hydroperoxy arachidonic acid. The PGI(2) activity of the supernates was destroyed by boiling or acidification. Inhibition of thrombin with diisopropylfluoro-phosphate, and of trypsin with soybean trypsin inhibitor, abolished the stimulation of PGI(2) production by these enzymes. Production of thrombin at a site of vascular injury could, by stimulating PGI(2) synthesis by endothelial cells adjacent to the injured area, limit the number of platelets involved in the primary hemostatic response and help to localize thrombus formation.     

Cholesterol enhances phospholipid-dependent activated protein C anticoagulant activity

The influence of cholesterol on activated protein C (APC) anticoagulant activity in plasma and on factor Va inactivation was investigated. Anticoagulant and procoagulant activities of phosphatidylcholine/phosphatidylserine (PC/PS) vesicles containing cholesterol were assessed in the presence and absence of APC using factor Xa-1-stage clotting and factor Va inactivation assays. Cholesterol at approximate physiological membrane levels (30%) in PC/PS (60%/10% w/w) vesicles prolonged the factor Xa-1-stage clotting time dose-dependently in the presence of APC but not in the absence of APC. APC-mediated cleavage of purified recombinant factor Va variants that were modified at specific APC cleavage sites (Q306/Q679-factor Va; Q506/Q679-factor Va) was studied to define the effects of cholesterol on APC cleavage at R506 and R306. When compared to control PC/PS vesicles, cholesterol in PC/PS vesicles enhanced factor Va inactivation and the rate of APC cleavage at both R506 and R306. Cholesterol also enhanced APC cleavage rates at R306 in the presence of the APC cofactor, protein S. In summary, APC anticoagulant activity in plasma and factor Va inactivation as a result of cleavages at R506 and R306 by APC is markedly enhanced by cholesterol in phospholipid vesicles. These results suggest that cholesterol in a membrane surface may selectively enhance APC activities.     

Thrombin-induced exposure and prostacyclin inhibition of the receptor for factor VIII/von Willebrand factor on human platelets.

The receptor for Factor VIII/von Willebrand factor (F. VIIIVWF) is readily available on circulating platelets. We have found that the stimulation of platelets with traces of thrombin at concentrations that are generated physiologically (0.008 U-0.05 U/ml) induced concentration-dependent binding of 125I-labeled F. VIIIVWF in a steady-state system. The binding induced by thrombin was specific because it was inhibited by a 100-fold molar excess of unlabeled F. VIIIVWF factor, by rabbit monospecific antibody against Factor VIII, and was not inhibited by an excess of fibrinogen or fibronectin. Binding induced by thrombin required metabolically active platelets, in contrast to a system with ristocetin that also prompted binding to glutaraldehyde-treated platelets. The thrombin effects on binding of 125I-F. VIIIVWF was not observed when platelets were washed with EDTA-containing buffers; EDTA and EGTA both inhibited thrombin-induced binding. Platelet membrane glycoproteins were required because enzymatic stripping od them from the platelet surface with chymotrypsin reduced binding 2.5-5.0-fold. Prostacyclin, in the concentration range of 1 to 50 nM, had two distinct effects on the receptor for F. VIIIWVF: (a) it prevented exposure of this receptor when added 10 min before thrombin, and (b) it reversed the binding of 125I-F. VIIIVWF to the platelet receptor when added 30 min after thrombin and the ligand, ie., when binding was at equilibrium. The dual effect of prostacyclin on the receptor for F. VIIIVWF was reproduced by dibutyryl cyclic AMP.     

Inactivation of human factor VIII by activated protein C. Cofactor activity of protein S and protective effect of von Willebrand factor.

Activated protein C (APC) acts as a potent anticoagulant enzyme by inactivating Factor V and Factor VIII. In this study, protein S was shown to increase the inactivation of purified Factor VIII by APC ninefold. The reaction rate was saturated with respect to the concentration of protein S when protein S was present in a 10-fold molar excess over APC. The heavy chain of Factor VIII was cleaved by APC and protein S did not alter the degradation pattern. Factor VIII circulates in a complex with the adhesive protein von Willebrand factor. When purified Factor VIII was recombined with von Willebrand factor, the inactivation of Factor VIII by APC proceeded at a 10-20-fold slower rate as compared with Factor VIII in the absence of von Willebrand factor. Protein S had no effect on the inactivation of the Factor VIII-von Willebrand factor complex by APC. After treatment of this complex with thrombin, however, the actions of APC and protein S towards Factor VIII were completely restored. In hemophilia A plasma, purified Factor VIII associated with endogenous von Willebrand factor, resulting in a complete protection against APC (4 nM). By mixing hemophilic plasma with plasma from a patient with severe von Willebrand's disease, we could vary the amount of von Willebrand factor. 1 U of von Willebrand factor was needed to provide protection of 1 U Factor VIII. Also in plasma from patients with the IIA-type variant of von Willebrand's disease, Factor VIII was protected. In von Willebrand's disease plasma, which was depleted of protein S, APC did not inactivate Factor VIII. These results indicate that protein S serves as a cofactor in the inactivation of Factor VIII and Factor VIIIa by APC and that von Willebrand factor can regulate the action of these two anticoagulant proteins.     

Inhibition by human thrombomodulin of factor Xa-mediated cleavage of prothrombin.

Human thrombomodulin significantly inhibited the rate of prothrombin conversion to thrombin by Factor Xa in the presence of phospholipid or platelets, calcium, and Factor Va. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of 125I-prothrombin activation revealed that thrombomodulin reduced the rate of prothrombin activation but did not alter the cleavage pattern. The inhibition was reversed by the inclusion of a highly specific rabbit antithrombomodulin antibody. If thrombomodulin was replaced by hirudin, the rate of thrombin generation was not decreased excluding the possibility that the inhibition by thrombomodulin was secondary to the binding of small amounts of thrombin formed early in the reaction and the prevention of feedback breakdown of prothrombin by thrombin. The inhibitory activity of thrombomodulin was overcome by increasing the concentration of Factor Xa and specific, saturable binding of thrombomodulin to Factor Xa was demonstrated. These results indicate that thrombomodulin binds to Factor Xa and thereby inhibits the activity of the prothrombinase complex.     

Evidence of normal functional levels of activated protein C inhibitor in combined Factor V/VIII deficiency disease.

Human activated protein C (APC) is a plasma serine protease that possesses amidolytic and anticoagulant activity. The rate at which the amidolytic and anticoagulant activity of APC was neutralized in normal plasma was essentially identical to that observed in plasma obtained from four individuals with combined Factor V/VIII deficiency disease. Incubation of radioiodinated APC with either normal human plasma or the combined Factor V/VIII-deficient plasmas resulted in the formation of a stable complex (Mr = 96,000) of the enzyme and a plasma protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Pretreatment of the radiolabeled APC with diisopropyl fluorophosphate prevented the formation of the enzyme-protein complex. On the basis of its ability to form a complex with radiolabeled APC, the APC-binding protein was purified to homogeneity from normal human plasma by ammonium sulfate fractionation, heparin-agarose chromatography, and QAE-Sephadex A-50 chromatography. The APC-binding protein (Mr = 54,000) is a glycoprotein, and possesses an amino-terminal sequence of Gly-Arg-Thr-Cys-Pro-Lys-Pro-Asp. The amino-terminal sequence of the APC-binding protein exhibited considerable homology with bovine colostrum inhibitor and pancreatic trypsin inhibitor, but no apparent sequence homology with the plasma serine protease inhibitors. Affinity-purified antibody against APC-binding protein immunoprecipitated a complex of radiolabeled APC and native APC-binding protein from normal human plasma. Complex formation was virtually eliminated in plasma immunodepleted of the APC-binding protein. Quantitative electroimmunoassay indicated essentially equal levels of APC-binding protein antigen in normal plasma compared with plasma from four patients with combined Factor V/VIII deficiency disease.     

Monoclonal immunoglobulin M lambda coagulation inhibitor with phospholipid specificity. Mechanism of a lupus anticoagulant.

Prolongation of all phospholipid-dependent coagulation tests was found in a patient with macroglobulinemia, despite absence of bleeding manifestations. The purified monoclonal IgM lambda protein and its Fabmu tryptic fragment induced similar changes in normal plasma. Patient IgM and Fabmu completely inhibited Ca++-dependent binding of radiolabeled prothrombin and Factor X to mixed phospholipid micelles. The patient's IgM lambda paraprotein reacted with phosphatidylserine and, to a lesser extent, with phosphatidylinositol and phosphatidic acid, but not with phosphatidylcholine or phosphatidylethanolamine. Prior incubation of phospholipid with patient Fabmu blocked the positive reactions. Substitution of washed platelets for phospholipid led to normalization of patient coagulation tests and corrected all abnormalities produced in normal plasma by patient IgM. Furthermore, binding of 125I-Factor Xa to thrombin-treated platelets was entirely normal in the presence of patient IgM. These studies support the concept that platelets, rather than phospholipid micelles, are the primary locus of prothrombin and Factor X activation in normal hemostasis.     

Characterization of an acquired inhibitor to coagulation factor V. Antibody binding to the second C-type domain of factor V inhibits the binding of factor V to phosphatidylserine and neutralizes procoagulant activity.

Coagulation Factor V is an essential component of the prothrombinase complex, which activates the zymogen prothrombin to thrombin. A patient was described who developed a Factor V inhibitor that neutralized the procoagulant activity of Factor V and resulted in a fatal hemorrhagic diathesis (Coots, M. C., A. F. Muhleman, and H. I. Glueck. 1978. Am. J. Hematol. 4:193-206). This inhibitor was shown to be an IgG antibody that bound to the light chain of Factor V. Using a series of light chain deletion mutants, we have found that this antibody binds to the second C-type domain of the light chain. Both inhibitor IgG and Fab fragments rapidly neutralized the procoagulant activity of Factor Va, implying that the neutralization resulted from specific binding to the C2 domain. We have previously demonstrated that deletion of the C2 domain results in loss of procoagulant activity, as well as loss of phosphatidylserine-specific binding. Confirming these results, both inhibitor IgG and Fab fragments interfered with phosphatidylserine-specific binding of Factor V. Conversely, preincubation of Factor Va with procoagulant phospholipids protected the cofactor from inactivation by the inhibitor. Our results suggest that this inhibitor neutralizes the procoagulant activity of Factor Va by interfering with the C2-mediated interaction with phospholipid surfaces, thereby disrupting formation of the prothrombinase complex.